Viral vectors with modified tropism

ABSTRACT

The present invention relates to gene therapy. In particular, therapeutic agents, therapeutic gene products, and compositions are disclosed. Various systems and methods useful in targeting and delivering non-native nucleotide sequences to specific cells are disclosed, wherein virus-antibody-ligand conjugates are used to facilitate targeting and delivery.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. ProvisionalApplication Serial Nos. 60/040,782 and 60/065,265, filed Mar. 14, 1997and Nov. 10, 1997, respectively. The disclosures of these applicationsare incorporated herein in their entirety by reference thereto.

TECHNICAL FIELD

[0002] The present invention relates to gene therapy. In particular,therapeutic agents and methods useful in targeting and delivering genesmore efficiently to particular cells are disclosed, wherein re-targeted,tropism-modified viral vectors presenting ligand on the surface andincluding a nucleotide sequence encoding a therapeutic gene product areused to facilitate targeting and delivery.

BACKGROUND OF THE INVENTION

[0003] The primary impediment to the transfer of non-native or foreignDNA into mammalian cells is that the genetic material must betransported across multiple cellular barriers before it enters the hostcell nucleus and initiates gene expression. Previously establishedmethods have utilized artificial means to introduce DNA into the cellalthough these methods are associated with significant cell toxicity(Graham et al., Virology 52:456-467 (1973); Felgner et al, PNAS USA84:7413-7417 (1987)).

[0004] More recently, enhanced transfer of DNA conjugates into cells hasbeen achieved with adenovirus, a human DNA virus which readily infectsvarious cell types (Horwitz, Adenoviridae and their replication, inVirology, Fields and Knipe, eds., Raven Press, NY (1990) pp. 1679-1740).Since adenovirus efficiently disrupts the membranes of endocyticvesicles, co-internalization of the virus with the DNA conjugate allowsrapid transfer of the conjugate into the cell cytoplasm before it can besubjected to lysosomal degradation. The fact that adenovirus exhibitsselective tropism has also been exploited to reconstitute these cells invivo with the human cystic fibrosis transmembrane conductance regulator(CFTR) (Rosenfeld et al., Cell 68:143-155 (1992)) and the alpha1-antitrypsin genes (Rosenfeld et al., Science 252:431-434 (1991)).

[0005] A number of features make adenoviruses very attractive for genedelivery applications. Knowledge of the adenovirus genetic system isfairly extensive, including the viral life cycle, DNA replication,transcription and RNA processing, and regulation of virus geneexpression. In addition, the size of the adenovirus (Ad) genome allowsrelatively easy manipulation of the viral DNA while still having thecapacity for insertion of most cDNAs into the viral genome. Additionaladvantages of adenovirus vectors include their ability to infect bothdividing and nondividing cells efficiently, to induce high-level foreignprotein expression without replication or integration of the viralgenome, and to grow to high yields when propagated in an appropriatecomplementing cell line.

[0006] If a target tissue lacks sufficient levels of adenovirusattachment receptors to mediate viral adsorption, however, this may alsobe a barrier to efficient gene transfer. Infection by most virusesrequires viral attachment to its host cell receptor. Adenovirus attachesto its host cell receptor via its fiber protein (see, e.g., Wickham etal., Cell 73:309-319 (1993)).

[0007] The Ad fiber protein is a long, trimeric protein that protrudesfrom the surface of the virion. At the distal end of the fiber proteinis a knob-like C-terminus that interacts with an unidentified cellularreceptor present on HeLa and other epithelial-derived carcinoma celllines (see, e.g., Defer et al., J. Virol. 64:3661-3673 (1990)). Thereceptor, generally identified as FibR, is assumed to be expressed bycells that are the normal targets for adenovirus infection.

[0008] Thus, reduced gene delivery to certain tissues may well resultfrom a low expression of the adenovirus receptor (FibR). A lack offunctional receptors is thus likely to be directly correlated withdramatic reductions in gene transfer efficiency.

[0009] In general, adenoviral vectors possess the capacity for in vivogene transfer that are critical to effective gene therapy. Followingadministration of the adenovirus vector, three distinct, sequentialsteps are required for expression of the therapeutic gene in targetcells: (I) attachment of the adenovirus vector to specific receptors onthe surface of the target cell; (2) internalization of the virus; and(3) transfer of the gene to the nucleus where it can be expressed. Thus,any attempt to modify the tropism of an adenovirus vector—that is, itsnative ability to target its cognate receptor must reserve its abilityto perform these three functions efficiently.

[0010] Investigators have met with greater or lesser success in thisregard. For example, the methodology proposed by Krasnykh et al. (J.Virol. 70: 6839-46 (1996) generates recombinant adenovirus with chimericfibers having a fiber shaft from one Ad serotype and a knob fromanother, thereby altering the adenovirus' receptor recognition profile.(Also see Gall et al., J. Virol. 70:2116-2123 (1996), which describes anAd 5/7 capsid chimera.) However, such constructs would appear to havelimited utility, as they still rely on the less-than-ubiquitous (andless-than-efficient) Ad receptors for targeting. Moreover, Ad vectorsthat rely upon Ad receptors for targeting (and putative internalization)are not able to target as wide a variety of cells as one might wish, anddepending on the nature of the chimeric fiber, any alterations in itsconformation may have a negative impact on targeting and/or delivery.

[0011] Further, the modifications described in the aforementionedarticles do not alter viral tropism in a manner that enhances viraltargeting or increases trafficking to the nucleus, contrary to what isdisclosed herein. In addition, the art fails to disclose targeting anddelivery constructs and systems that achieve the unexpectedly high levelof “infectivity” and expression shown herein. Finally, the constructsand methods of the present invention successfully achieved delivery oftherapeutic agents to cells that are normally resistant toviral-mediated delivery.

[0012] In view of the aforementioned problems, the design andconstruction of the within-disclosed vectors and conjugates provides anovel and elegant solution, as described further herein. The use of therecombinant sequences and vectors of this invention to mediate thetransfer of foreign genes into recipient cells both in vitro and in vivoovercomes the limitations of the above-described gene transfer systems.This invention utilizes recombinant constructs which confer theadvantages of targeting via the fibroblast growth factor receptor uponadenovirus—in place of the adenovirus usual targeting via fiberprotein—and thus represents an improved method for gene therapy as wellas for therapeutic applications involving delivery of a gene.

BRIEF SUMMARY OF THE INVENTION

[0013] In contrast to the disadvantages of using intact adenovirus,modified adenovirus vectors requiring a helper plasmid or virus, orso-called replication-deficient adenovirus, the use of recombinantadenovirus-derived vectors according to the present invention providescertain advantages for gene delivery. In particular, adenoviral vectorshaving their native tropism modified or ablated, which are thenre-targeted via a targeting ligand, are disclosed herein as advantageousfor a variety of gene therapy applications.

[0014] The Ad-derived vectors of the present invention possesses all ofthe functional properties required for gene therapy including binding tospecific cell receptors and penetration of endocytic vesicles. Theyfurther include those in which all or part of the fiber head or tail isreplaced with—or conjugated to—a ligand-encoding gene. Use of thevectors and conjugates disclosed herein allows one to target a widevariety of cells and to deliver therapeutic agents—irrespective ofwhether those agents are proteins, polypeptides, nucleotide sequences,or some other molecular species—directly into specific target cells.

[0015] The presently-disclosed constructs, systems and methods afford alevel of flexibility in therapeutic approaches not seen with othersystems and methods. Therefore, the vectors, systems and methods of thepresent invention are ideal for use in a wide variety of therapeuticapplications.

[0016] Therefore, in one embodiment, the present invention provides atropism-modified adenoviral vector system that specifically targetscells expressing a preselected receptor, comprising an antibody orfragment thereof that binds an adenoviral capsid protein; a targetingligand that binds the preselected receptor; and an adenovirus containinga nucleic acid molecule that encodes a therapeutic gene product underthe control of a promoter; wherein the ligand is conjugated to theantibody or fragment thereof and wherein the antibody or fragmentthereof is bound to the adenovirus. In one variation, the ligand isconjugated to the antibody or fragment thereof as a fusion, e.g., afusion-sFv. In another variation, the promoter is a tissue-specificpromoter.

[0017] In another embodiment, a tropism-modified adenoviral vector isprovided wherein the targeting ligand is a polypeptide reactive with anFGF receptor. In one variation, the polypeptide reactive with an FGFreceptor is an antibody or fragment thereof. In another variation, theantibody is a single-chain antibody. In one alternative embodiment, theantibody is 11A8. In another, the polypeptide reactive with an FGFreceptor is selected from the group consisting of FGF-1, FGF-2, FGF-3,FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-11, FGF-13, FGF-14,FGF-15, and molecules having 20% or greater homology to any of theforegoing. In still another embodiment, the polypeptide reactive with anFGF receptor is FGF-2. Yet another variation provides that the targetingligand is selected from the group consisting of a polypeptide reactivewith a VEGF receptor, a polypeptide reactive with a PDGF receptor, and apolypeptide reactive with an EGF receptor.

[0018] In other variations of the disclosed invention, thesurface-presented ligand is a polypeptide reactive with a cell-surfacereceptor; growth factor receptors are one class of receptorscontemplated within the scope of the present invention. Another class ofreceptors contemplated within the scope of the invention includesreceptors for Her-2/neu or erbB2. Thus, a polypeptide reactive with acell-surface receptor according to the present invention includesantibodies or fragments thereof, including single-chain antibodies,which react with receptors for Her-2/neu or erbB2.

[0019] The present invention also discloses embodiments wherein thenative tropism of the viral vector is modified; in still otherembodiments, the native tropism of the viral vector is ablated. Invarious preferred embodiments, the vector is an adenoviral vector. Theadenoviral vector is readily selected from any of the adenovirusserotypes, as well.

[0020] In a further aspect of the present invention, a tropism-modifiedvector is disclosed wherein the therapeutic gene product is a cytocideor a prodrug. In one set of related embodiments, the cytocide is aribosome inactivating protein. In other variations, the gene product isthymidine kinase, cytosine deaminase, or nitroreductase.

[0021] According to various embodiments of the present invention, thetherapeutic gene product enhances cellular proliferation. In onevariation, the therapeutic gene product is a biologically active proteinor polypeptide that augments or complements an endogenous protein. Inanother variation, the therapeutic gene product enhances cellulardifferentiation. In still another variation, the therapeutic geneproduct is a molecule which enhances tissue repair or regeneration. Yetanother variation provides that the therapeutic gene product is amolecule which stimulates a protective immune response.

[0022] The present invention further discloses a variety ofpharmaceutical compositions. In one embodiment, a pharmaceuticalcomposition of the present invention comprises a physiologicallyacceptable buffer and a tropism-modified adenoviral vector presenting aligand on its surface, wherein the vector includes a nucleic acidmolecule encoding a therapeutic gene product under the control of apromoter. In one variation, the ligand is a polypeptide reactive with anFGF receptor. In various alternative embodiments, the polypeptidereactive with an FGF receptor is selected from the group consisting ofFGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-11,FGF-13, FGF-14, and FGF-15. In one preferred variation, the polypeptidereactive with an FGF receptor is FGF-2. In other embodiments, thepolypeptide reactive with an FGF receptor is an antibody or fragmentthereof. In one alternative variation, the antibody is a single-chainantibody. In another variation, the antibody is 11A8.

[0023] In other variations of the disclosed invention, thesurface-presented ligand is a polypeptide reactive with a cell-surfacereceptor; growth factor receptors are one class of receptorscontemplated within the scope of the present invention. Another class ofreceptors contemplated-within the scope of the invention includesreceptors for Her-2/neu or erbB2. Thus, a polypeptide reactive with acell-surface receptor according to the present invention includesantibodies or fragments thereof, including single-chain antibodies,which react with receptors for Her-2/neu or erbB2.

[0024] In various aspects of the present invention, the ligand isgenetically fused with an adenoviral capsid protein. In others, theligand is chemically conjugated to an adenoviral capsid protein. In onevariation, the ligand is conjugated to an antibody or fragment thereofthat binds a viral capsid protein. In another variation, the ligand isconjugated to the antibody or fragment thereof as a fusion, e.g., afusion-sc-F_(v).

[0025] Other variations contemplate that the therapeutic gene product isselected from the group consisting of protein, ribozyme and antisense.In one alternative embodiment, the therapeutic gene product is acytocide. Exemplary cytocides include ribosome-inactivating proteins. Inanother embodiment, the therapeutic gene product is a prodrug. Exemplaryprodrugs include thymidine kinase, cytosine deaminase, ornitroreductase. Other embodiments disclose a wide variety of therapeuticgene products, including products that replace or repair defective,improperly regulated, or nonfunctional genes. In various alternativeembodiments, therapeutic gene products within the context of the presentinvention stimulate wound healing, tissue repair, connective tissuegrowth, angiogenesis, or the amelioration of ischemia, to name a fewexamples. In other embodiments, therapeutic gene products treat,interfere with or block a disease process, such as hyperproliferation ofcells, tumor growth, metastasis, and the like.

[0026] Thus, the present invention also discloses a variety of treatmentmethods. In one embodiment, the invention contemplates a method oftreating tumors, comprising administering a pharmaceutical compositioncomprising a physiologically acceptable buffer and a tropism-modifiedadenoviral vector presenting a ligand on its surface, wherein the vectorincludes a nucleotide sequence encoding a therapeutic gene product underthe control of a promoter, wherein the therapeutic gene product isselected from the group consisting of E-cadherin, BGP, Rb, p53,CDKN2/P16/MTS1, PTEN/MMAC1, APC, p331NG1, Smad4, maspin, VHL, WT1, Men1,NF2, MXI1, and FHIT. The invention also provides methods of treatingischemia, comprising administering a pharmaceutical compositioncomprising a physiologically acceptable buffer and a tropism-modifiedadenoviral vector presenting a ligand on its surface, wherein the vectorincludes a nucleotide sequence encoding a therapeutic gene product underthe control of a promoter, wherein the therapeutic gene product isselected from the group consisting of IGF, TGFβ1, TGFβ2, TGFβ3, HGF,VEGF 121, VEGF 165, FGF1, FGF2, FGF4, FGF5, PDGF-A, and PDGF-B.

[0027] In still other variations, the invention provides methods oftreating connective tissue injury, comprising administering apharmaceutical composition comprising a physiologically acceptablebuffer and a tropism-modified adenoviral vector presenting a ligand onits surface, wherein the vector includes a nucleotide sequence encodinga therapeutic gene product under the control of a promoter, wherein thetherapeutic gene product is selected from the group consisting of PTH,BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8, BMP10, BMP 11, mammalianBMP, and Xenopus BMP. An alternative method comprises the administrationof a pharmaceutical composition comprising a physiologically acceptablebuffer and a tropism-modified adenoviral vector presenting a ligand onits surface, wherein the vector includes a nucleotide sequence encodinga therapeutic gene product under the control of a promoter, wherein thetherapeutic gene product is selected from the group consisting of BovineVEGF, VEGF, VEGF-B, VEGF-C, Angiopoietin-1, Angiogenin, IGF-1, IGF-II,HGF, PDGF A, PDGF B, TGFB 1, TGFB2, and TGFB3.

[0028] The present invention also discloses various methods of treatingmalignancies, including cancer. In one such embodiment, a method oftreating cancer is disclosed, comprising contacting the cancer cellswith a pharmaceutical composition comprising a physiologicallyacceptable buffer and a tropism-modified adenoviral vector presenting aligand on its surface, wherein the vector includes a nucleotide sequenceencoding a therapeutic gene product under the control of a promoter,wherein the therapeutic gene product is selected from the groupconsisting of HSVTK, VZVTK, nitroreductase, and cytosine deaminase; andcontacting the cancer cells with a substrate. In various embodiments,the substrate is a molecule that is acted upon to produce a moleculethat is cytotoxic or cytostatic to the cancer cells.

[0029] In the various disclosed methods, the ligand is a polypeptidereactive with a specific cellular receptor; various polypeptides usefulin this regard are recited hereinabove. In various preferredembodiments, the receptor is an FGF receptor. In one variation, thepolypeptide reactive with an FGF receptor is FGF-2. In other variations,the polypeptide reactive with an FGF receptor is selected from the groupconsisting of FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8,FGF-9, FGF-11, FGF-13, FGF-14, and FGF-15.

[0030] In one alternative embodiment, the ligand is an antibody or afragment thereof. In another embodiment, the antibody is a single-chainantibody. In yet another variation, the ligand is conjugated to anantibody or fragment thereof that binds a viral capsid protein. Invarious embodiments, the viral capsid protein is adenovirus fiberprotein—for example, an adenovirus knob protein. In yet otherembodiments, the ligand may be chemically conjugated to a protein on thesurface of a viral vector or may be attached to the capsid as acomponent of a fusion protein.

[0031] In various methods disclosed herein, the therapeutic gene productis selected from the group consisting of protein, ribozyme andantisense. In one alternative embodiment, the therapeutic gene productis a cytocide. Exemplary cytocides include ribosome-inactivatingproteins. In another embodiment, the therapeutic gene product is aprodrug. Exemplary prodrugs include thymidine kinase, nitroreductase,and cytosine deaminase. Other embodiments disclose a wide variety oftherapeutic gene products, including products that replace or repairdefective, improperly regulated, or nonfunctional genes. In variousalternative embodiments, therapeutic gene products within the context ofthe present invention stimulate wound healing, tissue repair, connectivetissue growth, angiogenesis, or the amelioration of ischemia, to name afew examples. In other embodiments, therapeutic gene products treat,interfere with or block a disease process, such as hyperproliferation ofcells, tumor growth, metastasis, and the like.

[0032] These and other aspects of the present invention will becomeevident upon reference to the following detailed description andattached drawings. In addition, various articles and documents arereferenced herein to provide further details regarding variousprocedures, compositions, molecules, and the like. It is expressly to beunderstood that the disclosures of all publications referred to hereinare incorporated by reference in their entirety as though fully setforth herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1 shows a comparison of AdCMV-Luc transduction for four KScell lines. KS cells were incubated with recombinant adenovirusexpressing luciferase in the absence or presence of a Fab fragmentblocking adenoviral knob-mediated infection. Experiments were performedin triplicate. Relative light units (RLU) are shown on the verticalaxis; across the horizontal axis, the following cell lines areindicated: KSY-1; RW376; KS-SLK; and CVU-1. The open (colorless) barrepresents AdCMV-Luc, while the closed (dark) bar represents AdCMV-Luc+anti-knob Fab.

[0034]FIG. 2 shows the enhanced AdCMV-Luc infectivity of KS cell linesby Fab-FGF2 conjugate. The enhanced infectivity of the Ad-conjugatecomplex was assessed in the presence and absence of anti-FGF2 antisera.Relative light units (RLU) are plotted on the vertical axis, while therelevant KS cell lines—KSY-1, RW376, KS-SLK, and CVU-1—are indicated onthe horizontal axis. The closed bars represent AdCMV-Luc; stippled barsrepresent AdCMV-Luc +Fab-FGF2; and the open (colorless) bars representAdCMV-Luc +Fab-FGF2+anti-FGF2 antisera.

[0035]FIG. 3 illustrates enhanced AdCMVHSVtk/GCV cell killing in KSY-1and KS-SLK cells by Fab-FGF2 conjugate. The effect of GCV onAdCMVHStk-transfected cells was assessed in the presence or absence ofthe conjugate and expressed as a percentage of cells surviving comparedto the cell not exposed to GCV (i.e., −GCV). Viable cells in duplicatewells were counted, in triplicate, after trypan blue exclusion. On thevertical axis, the % of cells surviving is shown, in both FIGS. 3A and3B. In FIG. 3A, KSY-1 cells transfected with AdCMVHSVtk or AdCMVHSVtk+Fab-FGF2 are identified on the horizontal axis. In FIG. 3B, KS-SLKcells transfected with AdCMVHSVtk or AdCMVHSVtk +Fab-FGF2 are identifiedon the horizontal axis.

[0036]FIG. 4 illustrates a schema for the synthesis and purification ofthe Fab-FGF2 conjugate. It should be expressly understood that thisschema may be applied to the synthesis and purification of anyFab-ligand conjugate and is thus not limited to the one illustrated.

[0037]FIG. 5 shows the results of SDS-PAGE of Fab-FGF2 undernon-reducing conditions. Equal amounts (2 ug) of FGF2 (lane 2), Fab(lane 3), or Fab-FGF2 (lane 4) were applied to the gel and compared tothe molecular weight standards (lane 1, in thousands) by staining withCoomassie blue. In FIG. 5B, Western blot analysis of Fab-FGF2 conjugateis shown. The protein was transferred to a nitrocellulose membrane,probed with an anti-FGF2 rabbit polyclonal antibody and then with¹²⁵I-Protein A and visualized by autoradiography. A band was observedfor FGF2 (lane 5) and for the Fab-FGF2 conjugate (lane 7), but not forFab antibody alone (lane 6).

[0038]FIG. 6 shows functional validation of the Fab-FGF2 conjugate. InFIG. 6A, stimulation of endothelial cell proliferation by FGF2 and theFab-FGF2 conjugate is shown. Bovine aortic endothelial cells weretreated with various concentrations of FGF2 or Fab-FGF2 (30 pg/mL to 6ng/mL) and the cell number determined. Cell count (×1000) is plotted onthe vertical axis, while pg/mL are plotted on the horizontal axis. Opencircles represent FGF2, while closed circles represent Fab-FGF2.

[0039] In FIG. 6B, Fab-FGF2 binding to Ad5 knob in an ELISA isillustrated. Recombinant Ad5 knob was probed with either Fab-FGF2, ablank control, or FGF2. As a control, Fab-FGF2 was added to plates thatdid not contain Ad5 knob. Absorbance is plotted on the vertical axis,while the following are shown on the horizontal axis of the bar graph,proceeding from left to right: No knob +Fab-FGF2; Knob +Fab-FGF2; Knobalone; and Knob +FGF2.

[0040]FIG. 7 illustrates the results of in vitro infection of a panel ofcell lines using the Fab-FGF2 conjugate. In FIG. 7A, inhibition ofluciferase expression by the Fab is shown. The four cell lines wereinfected with either the AdCMVLuc or the AdCMVLuc premixed with the Fabas described in the text. The data are expressed as a percentage of theluciferase expression when AdCMVLuc alone was used for each cell line.Percentage is plotted on the vertical axis; cell lines 3T3, PANC-1,SKOV3.1p1, and D54 MG are illustrated along the horizontal axis. Openbars represent AdCMVLuc, while closed bars represent AdCMVLuc+Fab.

[0041] In FIG. 7B, luciferase expression in the four cell lines wheninfected with either AdCMVLuc or the AdCMVLuc-Fab-FGF2 conjugate isshown. The bars illustrate the luciferase expression in relative lightunits (RLU) per microgram of protein and represent triplicatemeasurements±standard deviation. RLU/ug of protein is plotted on thevertical axis. On the horizontal axis, cell lines 3T3, PANC-1,SKOV3.1p1, and D54 MG are illustrated. Closed bars represent AdCMVLuc,while cross-hatched bars represent AdCMVLuc +Fab-FGF2.

[0042] In FIG. 7C, inhibition of luciferase expression by the anti-FGF2antibody is shown. The four cell lines were infected with eitherAdCMVLuc premixed with the Fab-FGF2 conjugate or AdCMVLuc premixed withthe Fab-FGF2 conjugate and the anti-FGF2 antibody as described above.The data are expressed as a percentage of the luciferase expression seenwhen the ADCMVLuc-Fab-FGF2 complex was used for each cell line.Percentages are plotted on the vertical axis; cell lines 3T3, PANC-1,SKOV3.1p1, and D54 MG are illustrated along the horizontal axis. Lightlyshaded bars represent AdCMVLuc +Fab-FGF2, while the dark, closed barsrepresent AdCMVLuc +Fab-FGF2+anti-FGF2 Ab.

[0043] FIGS. 8A-C illustrate the expression of β-galactosidase in theliver of mice after treatment with Adβgal or FGF2-Adβgal. In Fig. A, noXgal stained cells in the liver of C57B1/6 mice treated with excipientare seen. In FIG. 8B, numerous Xgal stained hepatocytes are present inthe liver of C57B1/6 mice treated with Adβgal at a dosage of 2×10¹⁰ pfuper mouse, i.v. In FIG. 8C, treatment with FGF2-Adβgal at 2×10¹⁰ pfu permouse, i.v. transduces very few hepatocytes.

[0044]FIG. 9 shows the serum transaminase and alkaline phosphataselevels in mice treated with Adβgal or FGF2-Adβgal. Serum transaminases(AST, ALT) and alkaline phosphatase were measured on day 7 in C57B1/6mice after treatment with either excipient; Adβgal, 2×10e10 pfu, i.v.;or FGF2-Adβgal, 2×10e10 pfu, i.v.

[0045]FIGS. 10A and 10B illustrate the histopathology of the liver ofmice after treatment with Adβgal or FGF2-Adβgal. Hematoxylin and cosinstained paraffin sections of the liver of C57BI/6 mice treated witheither Adβgal, 2×10e10 pfu, i.v. (FIG. 10A); or FGF2-Adβgal, 2×10e10pfu, i.v. (FIG. 10B). Extensive hepatocellular necrosis and inflammatoryinfiltrate present in the liver of mice treated with Adβgal. There isnearly complete abrogation of hepatocellular necrosis in the liver ofmice treated with FGF-2Adβgal. Also, very little inflammatory infiltrateis observed.

[0046]FIG. 11 shows a survival analysis of tumor bearing mice treatedwith either Adtk or FGF2-Adtk. B16 melanoma cells were treated ex vivofor one hour with either Adtk or FGF2-Adtk and then implantedintraperitoneally into BDF1 mice at 2×10e6 cells per mouse. Mice werethen treated with either ganciclovir (GCV) or H₂O (as a control) for 14days, i.p. Survival of tumor bearing mice treated with FGF2-Adtk andthen administered ganciclovir was prolonged; such mice have astatistically prolonged survival compared to all other groups (p<0.001).

[0047]FIG. 12 illustrates the enhancement of Ad-mediated gene deliveryby the Fab-FGF2 conjugate. AdCMVLuc (5×10⁷ pfu) as preincubated with theoptimal dose of the Fab fragment (1.44 pg) or Fab-FGF2 conjugate (1.94μg) in 20 μL HBS for 30 min at room temperature. The vector or vectorcomplexes were then diluted in DMEM/F-12+2% FCS and 24,000 SKOV3.1p1cells in 24-well plates were infected at an MOI of 50 pfu/cell.Inhibition experiments were performed by adding a polyclonal anti-FGF2antibody (Sigma, St. Louis, Mo.) to the Ad CMVLuc-Fab-FGF2 complex priorto infection. Cell lysates were assayed for luciferase activity 24 hourspost-infection. The protein concentration of the lysates was determinedto permit normalization of the data, which are expressed as relativelight units (RLU) per microgram of cellular protein. Results are themean±SD of triplicate experiments.

[0048]FIG. 13 illustrates the results of FGF2-enhancement of Ad-mediatedexpression of the HSV-TK gene, which augments therapeutic benefit in asurvival experiment. A total of 95 female SCID mice aged 6-8 weeks wereinoculated intraperitoneally with 2×107 SKOV3.1p1 cells on day 0. On day5, some mice were injected intraperitoneally with 2×108 or 2×109 pfu ofAdCMVHSV-TK alone or AdCMVHSV-TK complexed with FGF2 (n=20 mice pergroup). Forty-eight hours later, half of the mice in each group (n=10)were treated daily with an intraperitoneal injection of ganciclovir (50mg/kg bodyweight) for 14 days. Control groups consisted of mice whichreceived no virus or GCV (n=5) or mice which were treated with GCV only(n=10). The mice were monitored daily for survival. The percentage ofanimals surviving is plotted against the number of days post tumor cellinoculation.

[0049]FIG. 14 illustrates antibody responses at day 21 following asingle injection of excipient, adenovirus or FGF-Fab:Ad conjugate.Optical density (O.D.)×10³ is plotted on the vertical axis, while datapoints corresponding to single injections of excipient, Ad, orFGF-Fab:Ad conjugate are identified on the horizontal axis. Opensquares, circles and diamonds correspond to anti-adenovirus proteinresponses, while closed squares, circles and diamonds correspond toanti-knob protein responses.

[0050]FIG. 15A shows that FGF2 retargeting of adenoviral infectionresults in increased transgene expression. HUVECs were infected withAdCMVLuc alone, AdCMVLuc+Fab, or AdCMVLuc+Fab-FGF2, and luciferaseexpression was measured 24 hours later. A representative graph of 3separate experiments is shown. Data is mean+/−SD. *p<0.001 vs. Ad alone.FIG. 15B shows the blocking of FGF2 retargeting. HUVECs were infectedwith AdCMVLuc +Fab-FGF2 alone or blocked with an anti-FGF antibody,heparin or excess free FGF. * p<0.001 vs. Ad⁺ Fab-FGF2. FIG. 15C showsthat enhanced gene expression is seen with FGF2. HUVECs infected with Adalone or in the presence of heparin or FGF2 at an equimolarconcentration to that used for Fab-FGF2 retargeting. FIG. 15Dillustrates the results of a tritiated binding assay. Tritiated AdCMVLucalone or following incubation with Fab, Fab-FGF2 or Fab-FGF2+antiFGFantibody was bound to cells at 4 degrees for 1 hr. Following washing,residual bound radioactivity (counts per minute, cpm) was measured in ascintillation counter. * p<0.001 vs. Ad alone, ** p<0.001 vs. Ad⁺Fab-FGF2.

[0051] FIGS. 16A-B illustrate that Fab-FGF2 retargeting enhances geneexpression in HCAECs and HASMCs. HCAECs (FIG. 16A) and HASMCs (FIG. 16B)were infected with AdCMVLuc alone, AdCMVLuc +Fab, or AdCMVLuc +Fab-FGF2,and luciferase expression was measured 24 hours later. A representativegraph of three experiments is shown. Data is mean+/−SD. * p<0.001

[0052]FIG. 17 describes the determination of the enhancing effect ofFGF2 retargeting at different viral doses. HUVECs were infected withAdCMVLuc at a dose of 10, 50 or 100 pfu/cell with or without Fab-FGF2retargeting. Luciferase assay was performed 24 hours later. Results aremean+/−SD.

[0053] FIGS. 18A-18G show the flow cytometry analysis of β-galactosidasetransduction. HUVECs infected at 10, 50 or 100 pfu/cell with AdCMVLacZor AdCMVLacZ +Fab-FGF. Determination of gene expression 48 hours laterby FDG staining and flow cytometry analysis. Representative profiles areshown, positive signal indicated by M2 gate.

[0054]FIG. 19 illustrates the reduction of FGF2 enhancement of genedelivery in quiescent cells. HUVECs were plated and maintained inculture for 1, 5 or 10 days. Cells were then infected with AdCMVLuc (50pfu/cell) or AdCMVLuc +Fab-FGF2 and luciferase assay was performed 24hours later. Data are expressed as the ratio of luciferase expression inthe cells infected using Fab-FGF2 retargeting, compared to correspondingcells plated at the same time and infected with AdCMVLuc alone. Barsrepresent mean±SD of three experiments. *p<0.01 vs. 1 day in culture.

[0055]FIG. 20 illustrates the successful retargeting of an Ad vectorlinked to a marker (Adβgal) using either FGF2 or 11A8-Fab and thesuccessful delivery of the marker sequence in HCT 116 cells. From leftto right, the shaded bars represent Adβgal; Fab; FGF2Fab, 30×; FGF2Fab,3×; 11A8Fab, 30×; and 11A8Fab, 3×. Molar excess of Ligand-Fab:KnobMonomer is indicated in the latter four categories. On the verticalaxis, mU βgal/mg protein is indicated. (Conditions: 25K; 72 hr; 300MOI.)

[0056]FIG. 21 illustrates the increased survival time seen in an in vivomurine tumor model when an Ad vector re-targeted with FGF2 anddelivering an intrabody payload is administered to SKOV3 tumor-bearingmice. Percent survival is plotted on the vertical axis;post-implantation survival (in days) is plotted on the horizontal axis.Closed circles represent mice receiving excipient alone (control);closed triangles represent mice receiving non-retargeted Ad deliveringHer-2/neu intrabody; and closed squares represent mice receivingFGF2-retargeted Ad delivering Her-2/neu intrabody. As indicated, N=β0;the dose administered was 1×10⁹ ADV or FGF-2 ADV.

DETAILED DESCRIPTION OF THE INVENTION

[0057] Many of the findings and results disclosed herein are quiteunexpected. For example, we have found that FGF retargeting of anadenovirus—i.e., altering the tropism of an adenovirus using afibroblast growth factor—significantly enhances targeting efficiency andnuclear trafficking of the Ad vector well above that seen when thevector retains its native Ad tropism. In addition, we observed that FGFretargeting increases the infectability of adenovirus in variouscells—e.g, cells expressing Kaposi's sarcoma—compared to the use ofnative Ad tropism alone. Interestingly, we found this to be true even inthose cell lines that were resistant to Ad infection.

[0058] Third, we found that the use of FGF retargeted vectors enhancespotency; FGF-retargeted vectors deliver and promote the expression of atherapeutic gene to more target cells and in each cell so targeted.Fourth, the vectors of the present invention are significantly lesstoxic to the liver and are less immunogenic than are other Ad vectors.Finally, we observed that retargeting the viral vector retargeted withFGF induces cytotoxicity to specific cell types when therapeutic genesequences (e.g. cytotoxic sequences, such as HSV-tk) are delivered; FGFretargeted vectors are thus able to transduce cells which are normallyinsensitive to Ad infection.

[0059] Thus, the FGF-retargeted vectors and related compositions andmethods of the present invention are unexpectedly and significantlysuperior to other gene therapy vectors, particularly viral vectors. Andwhile the retargeting of Ad vectors has been described herein asexemplary, it should be appreciated that other viral and non-viralvectors may benefit from the retargeting strategies disclosed herein.

[0060] Therefore, the present invention makes it feasible to engineerand produce novel constructs and vectors that are able to package anddeliver effective amounts of therapeutic agents or nucleic acidsequences encoding same for efficacious use in a variety of therapeuticapplications, without endangering the subject to whom they areadministered.

[0061] A. Definitions

[0062] Prior to setting forth the invention, it will be helpful to anunderstanding thereof to define certain terms used herein. The “aminoacids,” which occur in the various amino acid sequences appearingherein, are identified according to their well known three letter or oneletter abbreviations. The nucleotides, which occur in the various DNAfragments, are designated with the standard single letter designationsused routinely in the art.

[0063] As used herein, to “bind to a receptor” refers to the ability ofa ligand to specifically recognize and detectably bind to a receptor, asassayed by standard, e.g., in vitro, assays.

[0064] As used herein, a “conjugate” refers two or more molecules thatare linked together. The molecules may be conjugated directly or througha linker, such as a peptide, or they may be held together via ionic orother intermolecular forces. A conjugate may be produced by chemicalcoupling methods or by recombinant expression of chimeric DNA moleculesto produce fusion proteins.

[0065] A “cytocide-encoding agent” is a nucleic acid molecule thatencodes a product that results in cell death and generally acts byinhibiting protein synthesis. Such a product may act by cleaving rRNA orribonucleoprotein, inhibiting an elongation factor, cleaving mRNA, orother mechanism that reduces protein synthesis to a level such that thecell cannot survive. The product may be a protein, ribozyme, antisense,and the like. The nucleic acid molecule may contain additional elementsbesides the cytocide gene. Such elements include a promoter, enhancer,splice site, transcription terminator, poly(A) signal sequence,bacterial or mammalian origin of replication, selection marker, and thelike.

[0066] “Heparin-binding growth factor” refers to any member of a familyof heparin-binding growth factor proteins, in which at least one memberof the family binds heparin. Preferred growth factors in this regardinclude fibroblast growth factor (FGF), vascular endothelial growthfactor (VEGF), and heparin binding EGF-like factor (HBEGF). Such growthfactors encompass isoforms, peptide fragments derived from a familymember, splice variants, and single or multiple exons, some forms ofwhich may not bind heparin.

[0067] “Nucleic acid binding domain” (NABD) refers to a molecule,usually a protein, or peptide (but may also be a polycation) that bindsnucleic acids, such as DNA or RNA. An NABD may bind to single or doublestrands of RNA or DNA or mixed RNA/DNA hybrids. The nucleic acid bindingdomain may bind to a specific sequence or bind irrespective of thesequence.

[0068] As used herein, “nucleic acid” refers to RNA or DNA that isintended for internalization into a cell and includes, but is notlimited to, DNA encoding a therapeutic protein, a cytotoxic protein, aprodrug, a ribozyme, a deoxyribozyme, or antisense, the complement ofthese NAs, an antisense nucleic acid, and other such molecules.Reference to nucleic acids includes duplex DNA, single-stranded DNA, RNAin any form, including triplex, duplex or single-stranded RNA,anti-sense RNA, polynucleotides, oligonucleotides, single nucleotides,chimeras, and derivatives thereof. Nucleic acids may be composed of thewell-known deoxyribonucleotides and ribonucleotides (i.e., the basesadenosine, cytosine, guanine, thymidine, and uridine). As well, variousother nucleotide derivatives, non-phosphate backbones orphosphate-derived backbones may be used. For example, because normalphosphodiester oligonucleotides (referred to as PO oligonucleotides) aresensitive to DNA- and RNA-specific nucleases, oligonucleotides resistantto cleavage, such as those in which the phosphate group has been alteredto a phosphotriester, methylphosphonate, or phosphorothioate may be used(see U.S. Pat. No. 5,218,088).

[0069] As used herein, “receptor-binding internalized ligand” or“ligand” refers to any peptide, polypeptide, protein or non-protein,such as a peptidomimetic, that is capable of binding to a cell-surfacemolecule and internalization. Within the context of this invention, thereceptor-binding internalized ligand is conjugated to a nucleic acidbinding domain, either as a fusion protein or through chemicalconjugation, and is used to deliver a neuronal therapeutic-encodingagent to a cell. In one aspect, the ligand is directly conjugated to anucleic acid molecule, which may be further complexed with a nucleicacid binding domain. Such ligands include growth factors, cytokines,antibodies, hormones, and the like.

[0070] As used herein, a “targeted agent” is a chemical agent that isusually a nucleic acid molecule, but that may also be a protein, acarbohydrate, a lipid or any other class of chemical agent that isinternally delivered to a cell by a receptor-binding internalizedligand, and upon internalization alters or affects cellular metabolism,growth, activity, viability or other property or characteristic of thecell.

[0071] As used herein, a “therapeutic nucleic acid” describes anynucleic acid molecule used in the context of the invention that effectsa treatment, generally by modifying gene transcription or translation.It includes, but is not limited to, the following types of nucleicacids: nucleic acids encoding a protein, antisense RNA, DNA intended toform triplex molecules, protein binding nucleic acids, and smallnucleotide molecules. A therapeutic nucleic acid may be used to effectgenetic therapy by serving as a replacement for a defective gene, or byencoding a therapeutic product, such as a tumor-suppressing agent,prodrug, proliferation enhancer, or cytocide, to name a few examples.The therapeutic nucleic acid may contain all or a portion of a gene andmay function by recombining with DNA already present in a cell, therebyreplacing or complementing a defective portion of a gene. It may alsoencode a portion of a protein and exert its effect by virtue ofco-suppression of a gene product.

[0072] As used herein, “operative linkage” or operative association oftwo nucleotide sequences refers to the functional relationship betweensuch sequences. Nucleotide sequences include, but are not limited to,DNA encoding a product, DNA encoding a signal sequence, promoters,enhancers, transcriptional and translational stop sites, andpolyadenylation signals. For example, operative linkage of DNA encodinga therapeutic gene product to a promoter refers to the physical andfunctional relationship between the DNA and the promoter such thattranscription of the DNA is initiated from the promoter by an RNApolymerase that specifically recognizes, binds to, and transcribes theDNA.

[0073] As used herein, the phrase “polypeptide reactive with an FGFreceptor” refers to any polypeptide that specifically interacts with anFGF receptor (e.g. the high affinity FGF receptor), and is transportedinto the cell by virtue of its interaction with the FGF receptor.

[0074] As used herein, a “prodrug” is a compound that metabolizes orotherwise converts an inactive, nontoxic compound to a biologically,pharmaceutically, therapeutically, or toxic active form of the compound.A prodrug may also be a pharmaceutically inactive compound that ismodified upon administration to yield an active compound throughmetabolic or other processes. By virtue of knowledge of pharmacodynamicprocesses and drug metabolism in vivo, once a pharmaceutically activecompound is known inactive forms of the compound may be synthesized orisolated (see, e.g., Nogrady, Medicinal Chemistry A BiochemicalApproach, Oxford University Press, New York, pages 388-392, 1985).

[0075] As used herein, “receptor-binding internalized ligand” or“ligand” refers to any peptide, polypeptide, protein or non-protein,such as a peptidomimetic, that is capable of binding to a cell-surfacemolecule and internalizing. Within the context of this invention, thereceptor-binding internalized ligand may be conjugated to a viral capsidprotein, either as a fusion protein or through chemical conjugation,either directly or indirectly (e.g. via a bispecific antibody). By wayof further example, the ligand may be conjugated to the antibody orfragment thereof as a fusion, e.g., a fusion-sFv. Receptor-bindinginternalized ligands may thus be used to deliver therapeuticproduct-encoding agents to cells. Such ligands include growth factors,cytokines, antibodies, hormones, and the like.

[0076] A “wound site” as used herein is defined as any location in thehost that arises from traumatic tissue injury, or alternatively, fromtissue damage either induced by, or resulting from, surgical procedures.

[0077] B. Viral Vectors With Altered Tropism

[0078] Viruses—particularly adenoviruses—have not often been consideredto be ideal candidates for clinically-useful vectors, as their nativetropism causes them to be quite infective, as well as highly immunogenicand toxic. These principal difficulties, as well as others which relateto the virus' native tropism, have been encountered by others of skillin the art—and those difficulties have not been successfully overcome.

[0079] For example, the native forms of most (if not all) viral andretroviral vectors—that is, vectors possessing their native tropism—areunable to efficiently target potential host cells. This low targetingefficiency may be due to the failure of cells to express the appropriatereceptors or to express sufficient quantities of those receptors. Inmany instances, failure of the vector to escape the endosome to reachthe nucleus is a relevant factor, as well.

[0080] Merely altering the tropism of a vector, without more, may not besufficient to overcome the foregoing problems, however. For example,Douglas et al. described a method for ablating native Ad tropism whileconferring new tropism through the use of an anti-knob antibody whichwas conjugated to folic acid for targeting Ad to folatereceptor-positive cells (Douglas et al., Nature Biotech. 14:1574-1578(1996)). While the tropism-modified Ad was able to transduce folatereceptor-positive cells in vitro, the targeting efficiency was not asremarkable as that seen using the constructs and methods disclosedherein. Furthermore, delivery of the viral “payload” to the nucleus wasnot optimal.

[0081] Therefore, it is one goal of the present invention to design andconstruct viral constructs that have their native tropism modified(altered) or ablated (blocked). A further goal comprises modification ofthe virus in some fashion—e.g., genetically or immunologically—toprovide the virus with a new target. For example, preferred viralconstructs of the present invention possess the ability to targetparticular cell types.

[0082] Thus, as used herein, the term “tropism” refers to the movementor targeting of a viral vector (including viruses and viral particles)toward a receptor. Consistent with the foregoing, if a viral vector isdisplaying its native tropism, it is targeting its cognate receptor.

[0083] In the context of adenovirus (Ad), it tends to bind to integrinreceptors, which is believed to be required for subsequentinternalization of adenovirus into the host cell. Adenoviruses attach tohost-cell receptors via the penton fiber glycoprotein and enter cellsthrough the process of receptor-mediated endocytosis mediated by thepenton base. Ad apparently utilizes separate proteins for attachment andentry in a manner similar to enveloped human viruses.

[0084] The terms “retargeted,” “reprogrammed,” “tropism-modified,” or“altered tropism,” particularly as applied to viruses and viral vectors,are often used interchangeably herein and are meant to identify viralvectors whose endogenous (or native) tropism has been ablated ormodified. In one variation, the native tropism of the viral vector iseither unchanged, or it is modified, or even ablated; but the viralvector also includes a ligand which conveys an altered (i.e. non-native)tropism. As noted, such a modification may be partial—i.e., the viralvector retains at least a portion of its native tropism—or it may becomplete, whereby the native tropism of the viral vector is completelyablated.

[0085] The terms “tropism-modified,” “altered tropism,” and“reprogrammed,” as applied to viruses and viral vectors, also encompassviruses and vectors whose native tropism has been altered in some way(e.g., partially modified, or fully ablated) but which may not include aligand which confers a new tropism to the vector. And while the term“retargeted” is occasionally used to describe such vectors, it is moreappropriately used to describe vectors that do include a ligand whichconfers a new tropism to the vector. One should readily be able todiscern from the context of the description herein which variation isbeing described at any given point in the specification.

[0086] 1. Altering Viral Tropism

[0087] The development of viral vectors targeted to specific cell typeswill enhance their clinical application in the field of human genetherapy. To this end, several studies have focused on alteringadenovirus (Ad) tropism to direct the virus to cellular receptors otherthan the native cellular receptor by either expanding or limitingtropism. The former concept has been investigated in order to promotegene transfer in cells that are otherwise refractory towards Adinfection, while the latter approach targets Ad vectors to specific celltypes and limit gene transfer in non-target tissues. (See, e.g., Wickhamet al., Nature Biotech. 14:1570-1573 (1996); Wickham et al., J. Virol.70:6831-6838 (1996).)

[0088] While such studies employ different approaches for expanding Adtropism, they do not address the issue of whether it may be appropriateto modify native Ad tropism or to completely ablate native Ad tropism,which may well be necessary for effective clinical use in the context ofcancer gene therapy, for example. The present invention furtheraddresses the contexts in which one may wish to re-target an Ad vectorwithout altering its native tropism at all; when one may wish to modifyAd tropism by re-targeting it and by diminishing its native tropism; andwhen one may wish to re-target Ad and completely ablate its nativetropism.

[0089] We have discovered novel ligands that possess a remarkable andunrivaled ability to target specific cell types. Even more surprisingly,these same ligands are consistently trafficked to the cell nucleus insignificant quantity. As a result, these ligands are particularlydesirable for use in the targeting and delivery of a wide variety of“payloads” such as therapeutic nucleotide sequences encoding therapeuticgene products, and other molecules and agents that may impact nuclearand cellular functions.

[0090] For example, the use of FGF ligands and related moieties asefficient targeting and delivery agents is disclosed herein. When suchligands are linked in some fashion to a viral vector whose nativetropism has been blocked or otherwise ablated, targeting efficiencyincreases dramatically, as does trafficking of the ligand (and anythingconjugated or otherwise linked thereto) to the nucleus. Since FGFligands are associated with a wide variety of diseases and as theircognate receptors are expressed on a variety of cell types, such ligandsare ideal for use in the delivery of toxins in vitro and in vivo.

[0091] Moreover, when FGF ligands are used in conjunction with viralvectors—i.e., to confer a new tropism on such vectors—they are ideal foruse in the delivery of therapeutic nucleotide sequences, as well. When aviral vector possesses the ability to efficiently deliver geneticmaterial in vitro and in vivo—and Ad vectors are one such example—thecombination of FGF-related ligands and viral vectors with modifiedtropism is a powerful combination indeed.

[0092] As described in greater detail below, it has now been observedthat Ad-mediated gene transfer using a conjugate including anFGF-related ligand is greater than the level of gene transfer when Adalone is used. Other conjugates—e.g. the Fab-folate conjugate ofDouglas, et al. (Id., 1996) have not been able to facilitate Ad-mediatedgene transfer as efficiently as conjugates including FGF-relatedligands. Indeed, many such conjugates (including the Fab-folateconjugate) are not even able to reach the level achieved with Ad alone.

[0093] Irrespective of the explanation for the remarkable ability ofFGF-related ligands to achieve extremely efficient targeting anddelivery into specific cells, exploitation of this ability 1eAd5 to thedevelopment of viral vectors with new tropisms and enhancedgene-delivery potential.

[0094] 2. Exemplary Virus: Adenovirus

[0095] Since its discovery in 1953, the adenovirus has served as a modelfor molecular biology and cell transformation. The pentagonal capsomere(the penton) at the vertex of the adenovirus icosahedron consists of afiber projection, linked by non-covalent bonds to the penton base,anchored in the capsid. (See, e.g., Novelli and Boulanger, Virology185:365-376 (1991); Nermut, in The Adenoviruses, Ginsberg, ed., Plenum,N.Y., pp. 5-34 (1984); and Pettersson, in The Adenoviruses, Ginsberg,ed., Plenum, N.Y., pp. 205-207 (1984).)

[0096] Adenoviruses are nonenveloped, regular icosahedrons (20triangular surfaces and 12 vertices) that are about 65-80 nm in diameter(about 1400 angstroms (Å)). A structure, called fiber, projects fromeach of the vertices. The length of the fiber varies with the adenovirusserotype. The protein coat or capsid is composed of 252 subunits(capsomeres), of which 240 are hexons and 12 are pentons. Each of thepentons contains a penton base on the surface of the capsid and a fiberprojecting from the base, which is surrounded by five hexons. The namepenton is derived from these geometric relationships.

[0097] With regard to virion capsid polypeptides, most of the detailedstructural studies of adenovirus polypeptides have been performed for Adtypes 2 and 5. Many of the tropism-modified vectors disclosed herein arethus derived from the “better-characterized” Ad serotypes such as Ad 2,Ad 5, and Ad 21. However, due to the relative similarity and homologyamong the various human Ad serotypes, Ad vectors derived from any of theserotypes presently identified may be modified as disclosed herein.Human adenovirus serotypes from type 1 through 47 are currentlyavailable from the American Type Culture Collection (ATCC), Rockville,Md. and may thus be able to function effectively as vectors,particularly when modified according to the within-disclosed invention.

[0098] It should be understood that viral vectors of the presentinvention may be constructed using any appropriate and useful viralserotype. The invention is thus not limited to a particular serotype orserotypes.

[0099] All human adenoviruses examined to date encode a single fiberprotein with the exception of Ad40 and Ad41, which encode two fiberproteins and incorporate both polypeptides into their virions. Since thefiber interacts with a cellular receptor protein, these viruses mightrecognize two independent receptors. Fiber plays a crucial role inadenovirus infection by attaching the virus to a specific receptor onthe cell surface.

[0100] Adenovirus 2 (Ad2) DNA was the first adenovirus genome to becompletely sequenced; its sequence includes a total of 35,937 bp. Thesequence of Ad5 DNA was completed more recently; its sequence includes atotal of 35,935 bp. Portions of many other adenovirus genomes have alsobeen sequenced. It is presently understood that the upper packaginglimit for adenovirus virions is about 105% of the wild-type genomelength. (See, e.g., Bett et al., J. Virol. 67(10):5911-21 (1993).) Thus,for Ad2 and Ad5, this would be an upper packaging limit of about 38 kbof DNA.

[0101] While some prefer to use replication-defective Ad viral vectorsfor fear that replication-competent vectors raise safety issues, theviral vectors of the present invention may retain their ability toexpress the genome packaged within (i.e., they may retain theirinfectivity), but they do not act as infectious agents to the extentthat they cause disease in the subjects to whom they are administeredfor therapeutic purposes.

[0102] The Ad-derived viral vectors disclosed herein may be used totarget and deliver genes into specific cells by incorporating theattachment sequence for other receptors (such as FGF) onto the fiberprotein by recombinant DNA techniques or by immunological means, thusproducing chimeric molecules or conjugates. This should result in theability to target and deliver genes into a wide range of cell types withthe advantage of evading recognition by the host's immune system. Thewithin-disclosed targeting and delivery systems are also much moreefficient at targeting and delivery than are viral vectors utilizingtheir native tropism, as will be further illustrated below. Thus, thewithin-disclosed delivery systems and constructs provide for increasedflexibility in gene design to enable stable integration of molecules ofchoice into proliferating and nonproliferating cell types.

[0103] For example, published International App. No. WO95/26412, U.S.Pat. No. 5,543,328, and Krasnykh et al. (J. Virol. 70:6839-46 (1996)),the disclosures of which are incorporated by reference herein, describemodifications that may be made to the adenovirus fiber protein. Suchmodifications are useful in altering the targeting mechanism andspecificity of adenovirus and could readily be utilized in conjunctionwith the constructs of the present invention to target the novel viralvectors disclosed herein to different receptors and different cells.Moreover, modifications to fiber protein which alter its tropism maypermit greater control over the localization of viral vectors intherapeutic applications.

[0104] Similarly, incorporation of various structural proteins into celllines of the present invention, whether or not those proteins aremodified, is also contemplated by the present invention. Thus, forexample, modified penton base polypeptides such as those described inWickham et al. (J. Virol. 70:6831-8 (1996)) may have therapeutic utilitywhen used according to the within-disclosed methods.

[0105] C. Immunological Modification of Viral Tropism

[0106] One useful method of modifying viral tropism utilizesimmunological constructs. In various disclosed embodiments, it ispreferred to modify the virus' native tropism and to re-target the viralvector by linking it to a ligand—especially a receptor-binding,internalizing ligand. In other embodiments, it is preferable tocompletely ablate the native tropism of the virus and to replace it withan entirely new tropism. Any degree of modification of a virus' nativetropism—from partial modification through and including completeablation—may readily be accomplished using immunological means, asdescribed herein.

[0107] For example, one means of immunologically modifying a viralvector is via the construction of a bispecific antibody that binds to aviral capsid protein on one “end” and binds to a targeting moiety(ligand) on the other. In this way, a viral vector may be re-targetedvia the targeting ligand. If the capsid protein to which one portion ofthe antibody is bound happens to be the protein via which the virustypically binds and/or enters cells, then the virus' native tropism isaffected as well.

[0108] Other immunological means are also available. Construction offusion proteins—e.g., ligand-sFv fusions—is another method ofimmunologically modifying a viral vector. Similar to the foregoingexample, the ligand portion of the fusion confers a novel tropism uponthe vector, while the antibody portion links the ligand to the vector.As before, depending upon the function of the viral capsid protein towhich the antibody portion binds, attachment of the antibody may alsointerfere with or ablate the virus' native tropism.

[0109] Further, while these methodologies are rather less efficient, onemay readily generate multiple antibodies or fragments thereof—e.g.antibodies that modify or ablate the virus' native tropism, but which donot bind a ligand for retargeting purposes, and antibodies that bind aretargeting ligand and attach it to the viral capsid. One may alsogenerate anti-idiotype antibodies for similar purposes—i.e., to link newligands to viral vectors and/or to modify the virus' native tropism.

[0110] As one may imagine, however, when one is contemplating using theviral vectors for gene targeting and delivery purposes, the less “bulky”the construct is, the more readily it may be delivered to and into acell. Thus, constructs using antibody fragments which function to link atargeting ligand to the viral capsid and which simultaneously modify thevirus' native tropism are more ideal for use in gene therapyapplications.

[0111] 1. Anti-Viral Antibody Coniugated to Ligand

[0112] As discussed herein, growth factor receptor-bindingligands—particularly polypeptides reactive with an FGF receptor—areparticularly useful re-targeting agents.

[0113] Although any antibody that neutralizes or blocks a virus fromtargeting and binding a cell using its native tropism is contemplatedherein, adenoviral anti-knob antibodies and fragments thereof aredescribed herein as exemplary. Methods of preparing and using anti-knobantibodies and immunologically active fragments thereof are furtherdescribed in the Examples that follow. Similarly, methods of preparingand using antibody-ligand conjugates are also described.

[0114] Other methods of preparing anti-viral antibodies—and theantibodies so prepared—are available and are useful according to thewithin-disclosed methods, as well. For example, U.S. Pat. No. 5,521,291(the disclosures of which are incorporated by reference herein)describes a method of preparing a chimeric adenovirus having aheterologous epitope exposed in the exterior domain of its hexonprotein. Depending on the method used, the degree of modification ofnative viral tropism—which may range from no alteration all the way tocomplete ablation—may be adjusted as disclosed herein.

[0115] As discussed previously, adenoviral vectors possess the in vivogene transfer characteristics consistent with the within-disclosedtargeting and delivery applications that are critical to effective genetherapy. Following administration of the adenovirus vector, threedistinct, sequential steps are generally understood to be required forexpression of the therapeutic gene in target cells: (1) attachment ofthe adenovirus vector to specific receptors on the surface of the targetcell; (2) internalization of the virus; and (3) transfer of the gene tothe nucleus where it can be expressed. Thus, any attempt to modify thetropism of an adenovirus vector preferably preserves the vector'sability to perform these three functions efficiently. An understandingof the adenovirus entry pathway should also facilitate attempts tomodify the tropism of adenoviral vectors to permit the targeting ofspecific cell types.

[0116] For example, if the therapeutic goal is the modification of Adtropism for tumor cell-specific targeting, two linked requirements areinvolved. First, in order to restrict gene transfer exclusively to tumortargets ablation of endogenous viral tropism is preferably achieved.Second, a new binding specificity must be introduced into the adenoviralfiber protein to allow recognition of cell surface markerscharacterizing neoplastic cells. Ablation of endogenous adenoviraltropism with a neutralizing anti-knob monoclonal antibody (Mab), therebyallowing the introduction of novel tropism by conjugating acell-specific ligand to this Mab, is further discussed in Example 1below.

[0117] 2. Bi-Specific Antibodies

[0118] a. Background

[0119] In addition to the foregoing methodologies, it is also possibleto ablate endogenous adenoviral tropism by generating a bi-specificantibody that recognizes an Ad capsid protein (e.g. knob protein) aswell as the target cell-specific receptor. Polypeptides reactive with anFGF receptor are exemplary targeting ligands which are useful in thisregard, as discussed in greater detail below.

[0120] Our previous work with fused cDNAs encoding FGF2 and cytotoxinsestablished that FGF2 can serve as a vehicle to introduce DNA into cellswith specificity. Based on those studies, FGF2-anti-knob Fab complexeshave now been exploited for their ability to specifically target thewithin-disclosed adenoviral vectors to FGF receptor-bearing cells (seethe Examples below).

[0121] Recombinant adenoviral vectors have the ability to efficientlytransfer genes to a wide range of cell types in vitro and in vivo.Because of this, adenoviral vectors have been used in a number ofdifferent gene therapy approaches. However, adenovirus lacks the abilityto accomplish cell-specific targeted gene delivery because the tropismof the parent adenovirus is quite broad, permitting widespreadtransduction of various end organs after systemic in vivo delivery. Thisbroad tropism is based upon the fact that the cellular binding receptorfor adenovirus is ubiquitously expressed. It is this property of theadenovirus which undermines the potential utility of adenoviral vectorsas a candidate system for accomplishing the specific transduction ofdisseminated tumor cells. The lack of tumor-specific targeting ofadenoviral vectors would allow ectopic expression of the deliveredanti-cancer gene construct. Thus, despite the capacity of the adenoviralvector to accomplish high efficiency in vivo gene delivery via thevascular route, a means must be developed to redirect its tropismspecifically to tumor targets. This will require both the ablation ofthe endogenous viral tropism and the introduction of novel tropism.

[0122] Based on the above considerations, we hypothesize thatmodifications of adenoviral tropism can accomplish tumor cell-specifictransduction.

[0123] A number of studies have shown that retroviral cell-bindingactivity or tropism can be altered by modifications of the viralenvelope glycoprotein which interacts with specific receptors on thecell surface. One approach has involved the construction of“pseudotypes,” in which the retroviral genome is coated by the envelopeprotein of another virus (see, e.g., Weiss et al., Virology 76: 808-25(1977)). The host range of the pseudotyped particle is thus dictated bythe virus providing the envelope protein.

[0124] b. Construction of the Bifunctional scFv-FGF2 Fusion Protein

[0125] The neutralizing anti-knob mAb, 1D6.14, was generated asdescribed by Douglas et al. (Id. (1996)). The procedure may be describedessentially as follows. Fab fragments of the mAb are also prepared foruse in various constructs, as described herein. In addition to its usein the construction of fusion proteins, the mAb and fragments thereofare used to prepare FGF-Fab constructs as well (see Example 1, SectionA.4 below).

[0126] To develop a neutralizing anti-knob mAb, hybridomas weregenerated by standard techniques after immunization of mice with intactAd5 followed by two rounds of immunization with purified recombinant Ad5knob. BABL/c mice were immunized with Ad5, followed by two rounds ofimmunization with recombinant Ad5 knob, a gift from R. D. Gerard (Univ.of Texas). (Also see Henry et al., J. Virol. 68:5239-5246 (1994).)Sensitized lymphocytes were fused with P3-X63-Ag8.653 cells. Thereactivity of the hybridoma supernatants with trimeric Ad5 knob wasdetermined in an ELISA. The ability of the hybridoma supernatants toneutralize Ad5 infection was assayed by endpoint CPE.

[0127] On the basis of its high affinity binding to recombinant Ad5 knoband its ability to neutralize Ad5 infection of HeLa cells, one clone,designated 1D6.14, was chosen for further study. The selected mAb waspurified from ascites fluid by affinity chromatography using animmobilized protein A column.

[0128] Fab fragments were prepared and purified after digestion ofintact 1D6.14 with papain. Both the parent antibody and the Fab fragmentwere capable of neutralizing adenovirus infection in a dose-dependentmanner. (Douglas et al., Id. (1996)).

[0129] To generate the single chain ScFv, mRNA is isolated from thehybridoma and the ScFv was generated by splice site overlap extensionPCR using standard techniques (Miller, R. et al, manuscript inpreparation). This anti-knob scFv 01D6 will be employed to generate thebispecific retargeting fusion protein. This is accomplished by geneticinsertion of the FGF2 ligand by PCR based cloning into the 01D6 pET25 E.coli expression vector. Convenient restriction sites, Nco and Nhe 1,were added to the 5′ and 3′ respectively for ease of cloning.

[0130] The amplified heavy and light chain product was cloned into thepET25 expression vector (Novagen, Milwaukee, Wis.). The expressionvector, pET25, contains the promoter for T7 RNA polymerase, lacoperator, pelB leader sequence, the Nco 1 restriction site in frame withpelB, the HSVTag sequence and a His Tag sequence. The FGF2 is expressedas both N-terminal and C-terminal fusion protein.

[0131] In addition, flexible linkers may be added between the scFv andFGF2 to help favor proper protein folding. One strategy to clone theFGF2 downstream of the scFv is described below. To clone the ScFv-FGF2fusion protein, the stop codon is removed in the ScFv and human FGF2 iscloned downstream and in-frame with the ScFv. Oligos that may be used toremove the stop codon and add a restriction site in the ScFv include(5′-3′): A1 (sense): ATATAGAATTCTGTGACTACTGAGGACACAGCCAC and A2(antisense): ATATACATATGTTTTTTCAGCTCCAGCTTGGTCCC.

[0132] PCR amplification using these primers results in a 465 bpfragment. The PCR product has Eco R1 and Nde 1 sites a the 5′ and 3′ends. The amplified fragment is preferably digested with Eco R1 and Nde1, isolated by gel electrophoresis and purified using GENECLEAN (Bio101, Vista, Calif.). FGF2 is obtained by digestion with restrictionenzymes Nde 1 and Bam H1 from the pET 11a FGF2 expression vector(previously generated at PRIZM, San Diego, Calif.).

[0133] The remaining section of the ScFv is isolated by digestion of thepET25 ScFv with Nco 1 and Eco R1. The digested fragment is isolated byagarose gel and purified using GENECLEAN. The purified fragments areligated together with Nco 1 Bam H1 digested pET25 in a 4-way ligation.The ligation may be transformed in to Novablue (Novagen) and clonesevaluated for insertion of the fragments and further analyzed forcorrect restriction map. Glycerol stocks of clones with the correctrestriction map are also preferably generated. DNA is purified andsequenced for verification.

[0134] c. Expression, Purification and Evaluation of ScFv-FGF2 FusionProtein

[0135] Competent bacterial cells, BL21(DE3), are transformed with thepET25 pelB ScFv-FGF2 and pET25 pelB ScFv constructs. For expression, theplasmid transformed host cells are grown at 25-30° C. to an OD600 of 0.7and induced with IPTG. The culture is harvested 3-4 hours afterinduction. The suspension is centrifuged; the supernatant clarified andassayed for either ScFv-FGF2 or ScFv protein by ELISA. The ScFv andScFv-FGF2 fusion protein can be recognized by antiserum to both theheavy and light chains as well as to FGF2. A sample of the pellet andthe supernatant is analyzed by SDS-PAGE and Western analysis usingantibodies to FGF2 and heavy and light chains to determine thepercentage of fusion protein within each fraction. If the fusion proteinfractionates to the pellet then a refolding method such as lysis in 6 Mguanidine solution and gradual dialysis into non-denaturing buffer isattempted. Alternatively, the periplasmic proteins can be isolated byosmotic shock and assayed for fusion protein. Purification isaccomplished by either heparin chromatography or via the His Tag at theN-terminus of the fusion protein using metal chelate resin affinitychromatography. The purified fusion protein is tested for bindingactivity by ELISA using either FGF2 antibody coated plates or heavy andlight chain antibody coated plates and detected with alkalinephosphatase. We have initially designed this construct for expression inE. coli. However, if expression of the ScFv-FGF2 fusion protein isextremely low in bacteria, then the fusion protein can easily by excisedwith restriction enzymes and ligated into a mammalian expression vectorusing the Vh leader sequence.

[0136] To be useful in retargeting adenovirus, the recombinant fusionprotein must bind both the adenovirus knob as well as the cognatereceptor. Thus these proteins are analyzed for their knob bindingcapacity in an ELISA. To validate that they bind knob in its nativetrimeric form, each protein is used to probe boiled and unboiledrecombinant Ad5 knob in an immunoblot. Finally, a neutralizing assay isperformed using AdCMVLuc. To test the functionality of the receptorbinding domain, binding and internalization assays are performed onreceptor positive cells. The purified ScFv is tested for receptorbinding in endothelial cells as outlined in the preliminary results. Inaddition, binding and internalization studies are completed for thefusion protein. This is done by incubating receptor positive cells with[¹²⁵I] radiolabeled scFv-FGF2 fusion protein at 4° C. to preventinternalization. After removal of unbound protein, the amount ofradioactivity in released and cell-associated fractions is determined ina scintillation counter. Binding specificity is determined by includingunlabeled fusion protein as a competitor. Internalization of the fusionproteins is determined by preincubating receptor positive cells withlabelled fusion protein at 4° C., washing cells to remove unboundlabelled protein, warming to 37° C. for various time intervals to allowreceptor internalization. Following 2M salt extraction to remove surfacebound radiolabeled protein, cells are lysed and radioactivity isdetermined in the cell lysate. This analysis will determine the capacityof the recombinant bispecific fusion protein to bind Ad5 knob and FGFreceptors in the context of a fusion protein, as well as ablateendogenous adenovirus tropism. These molecules are then evaluated fortheir ability to target adenoviral infection via the FGF receptor asdescribed above.

[0137] D. Genetic or Chemical Modification of Viral Protein

[0138] Viral particles—e.g., an adenovirus protein—may alternatively bemodified at the molecular level. Thus, for example, a nucleotidesequence encoding a ligand molecule may be operatively linked forexpression to a viral nucleotide sequence—particularly to a sequenceencoding a structural protein.

[0139] Thus, one may construct fusion proteins and other modificationsof viral proteins. For example, the fiber protein of adenovirus may bemodified via attachment of a heterologous nucleotide sequence to theC-terminus of the gene encoding adenoviral fiber protein. Alternatively,one or more heterologous sequences may be inserted at an internalsite—i.e., within the viral fiber protein sequence.

[0140] Various methods of preparing such fusions are available in theart and are contemplated by the present invention. For example, U.S.Pat. No. 5,543,328, the disclosures of which are incorporated byreference herein, recites a method for removing all or a part ofadenovirus fiber protein and replacing the removed portion with a ligandthat is specific for a particular cellular receptor.

[0141] Similarly, Michael et al. proposed the addition of a shortpeptide ligand to Ad fiber protein via placing a sequence encoding theterminal decapeptide of gastrin releasing peptide (GRP) at the 3′ end ofthe coding sequence of the Ad5 fiber gene. (See Gene Therapy 2:660-668(1995), incorporated by reference herein.) Wickham et al. attached aheparin-binding domain to the Ad5 fiber protein and observed that the Advector displayed a new tropism. (See Nature Biotech. 14:1570-1573(1996), incorporated by reference herein.) Although the aforementionedconstructs may be useful as disclosed herein, none of them produced theunexpected, dramatic increases in targeting efficiency and nucleartrafficking obtained with the constructs of the present invention.

[0142] Finally, it should also be appreciated that viral proteins may bemodified via means that are not precisely “immunologic” or “genetic.”Modification of viral proteins via means other than those exemplifiedherein is fully within the scope of the present invention. For example,useful reprogrammed vectors of the present invention may undergochemical alteration of their native tropism, e.g., via chemicalinactivation of the virus, and they may subsequently be “reactivated” byanother molecule or process designed to retarget the viral vector.

[0143] Thus, heat inactivation is one method contemplated within thescope of chemical alterations which may be made to viral vectors of thepresent invention. Chemical alteration of the molecular moiety (e.g.fiber protein) in a manner that disrupts of ablates the vector'sendogenous tropism is also contemplated herein. Methods of alteringviral proteins via chemical means are known to those of skill in the artand may readily be ascertained in the relevant literature.

[0144] E. Ligands

[0145] As noted above, the present invention provides a variety ofmethods of “reprogramming” the tropism of a virus (or viral vector),including methods utilizing ligands such as FGF proteins, polypeptides,analogs or mimics to assist in re-targeting the vector. While certainligands are described as exemplary, it will be appreciated by those ofskill in the art that a wide variety of molecules may appropriately beused as ligands according to the within-disclosed methodologies. Thefollowing lists—while not exhaustive—will provide one with a betterunderstanding of the variety of ligands available for use tospecifically target preselected cells and to direct the vector,conjugate or complex with which the ligand is associated into thecell—and ideally, into the nucleus.

[0146] 1. Proteins That Bind to Cells and Internalize

[0147] The ligands may be produced by recombinant or other means inpreparation for attachment to viral (e.g. adenoviral) proteins. The DNAsequences and methods to obtain the sequences of these ligands are wellknown. (see GenBank). Based on the DNA sequences, the genes may besynthesized either synthetically (for small proteins), amplified fromcell genomic or cDNA, isolated from genomic or cDNA libraries and thelike. Restriction sites to facilitate cloning into the viral vector maybe incorporated, such as in primers for amplification.

[0148] Such molecules include, without limitation, proteins that bindcancer cells, endothelial cells, cardiovascular cells, cells in the eyeand the like. Such ligands include growth factors and cytokines. Manygrowth factors and families of growth factors share structural andfunctional features and may be used in the present invention. Familiesof growth factors include fibroblast growth factors FGF-1 throughFGF-15, and vascular endothelial growth factor (VEGF). Other growthfactors, such as PDGF (platelet-derived growth factor), TGF-α(transforming growth factor), TGF-β, HB-EGF, angiotensin, bombesis,erythropoietin, stem cell factor, M-CSF, G-CSF, GM-CSF, and endoglinalso bind to specific identified receptors on cell surfaces and may beused in the present invention. Cytokines, including interleukins, CSFs(colony stimulating factors), and interferons, have specific receptors,and may be used as described herein.

[0149] For example, ligands and ligand/receptor pairs includeurokinase/urokinase receptor (GenBank Accession Nos. X02760/X74309);α-1,3 fucosyl transferase, α1-antitrypsin/E-selectin (GenBank AccessionNos. M98825, D38257/M87862); P-selectin glycoprotein ligand, P-selectinligand/P-selectin (GenBank Accession Nos. U25955, U02297/L23088),VCAM11VLA-4 (GenBank Accession Nos. X53051/X16983); E9 antigen (Blann etal., Atherosclerosis 120:221, 1996)/TGFβ receptor; Fibronectin (GenBankAccession No. X02761); type I α1- collagen (GenBank Accession No.Z74615), type I β2-collagen (GenBank Accession No. Z74616), hyaluronicacid/CD44 (GenBank Accession No. M59040); CD40 ligand (GenBank AccessionNo. L07414)/CD40 (GenBank Accession No. M83312); ELF-3, LERTK-2 ligands(GenBank Accession Nos. L37361, U09304) for elk-1 (GenBank Accession No.M25269); VE-cadherin (GenBank Accession No. X79981); ligand forcatenins; ICAM-3 (GenBank Accession No. X69819) ligand for LFA-1, andvon Willebrand Factor (GenBank Accession No. X04385), fibrinogen andfibronectin (GenBank Accession No. X92461) ligands for α_(v)β₃ integrin(GenBank Accession Nos. U07375, L28832).

[0150] Other ligands include CSF-1 (GenBank Accession Nos. M11038,M37435); GM-CSF (GenBank Accession No. X03021); IFN-α (interferon)(GenBank Accession No. A02076; WO 8502862-A); IFN-γ (GenBank AccessionNo. A02137; WO 8502624-A); IL-1-α (interleukin 1 alpha) (GenBankAccession No. X02531, M15329); IL-1-β (interleukin 1 beta) (GenBankAccession No. X02532, M15330, M15840); IL-1 (GenBank Accession No.K02770, M54933, M38756); IL-2 (GenBank Accession No. A14844, A21785,X00695, X00200, X00201, X00202); IL-3 (GenBank Accession No. M14743,M20137); IL-4 (GenBank Accession No. M13982); IL-5 (GenBank AccessionNo. X04688, J03478); IL-6 (GenBank Accession No. Y00081, X04602, M54894,M38669, M14584); IL-7 (GenBank Accession No. J04156); IL-8 (GenBankAccession No. ZI 1686); IL-10 (GenBank Accession No. X78437, M57627);IL-11 (GenBank Accession No. M57765 M37006); IL-13 (GenBank AccessionNo. X69079, U10307); TNF-α (Tumor necrosis factor) (GenBank AccessionNo. A21522); TNF-β (GenBank Accession No. D12614); urokinase/urokinasereceptor (GenBank Accession Nos. X02760/X74309); α-1,3 fucosyltransferase, α1-antitrypsin/E-selectin (GenBank Accession Nos. M98825,D38257/M87862); P-selectin glycoprotein ligand, P-selectinligand/P-selectin (GenBank Accession Nos. U25955, U02297/L01574);VCAM11VLA-4 integrin receptor (GenBank Accession Nos. X53051/X16983 andL12002); E9 (Blann et al., Atherosclerosis 120:221, 1996)/TGFβ receptor;Fibronectin (GenBank Accession No. X02761); type I^(α1) collagen(GenBank Accession No. Z74615), type I β2-collagen (GenBank AccessionNo. Z74616), hyaluronic acid/CD44 (GenBank Accession No. M59040); CD40ligand (GenBank Accession No. L07414)/CD40 (GenBank Accession No.M83312); EFL-3, LERTK-2 ligands (GenBank Accession Nos. L37361, U09304)for elk-1 (GenBank Accession No. M25269); VE-cadherin (GenBank AccessionNo. X79981) ligand for catenins; ICAM-3 (GenBank Accession No. X69819)ligand for LFA-1, and von Willebrand Factor (GenBank Accession No.X04385), fibrinogen and fibronectin (GenBank Accession No. X92461ligands for α_(v)β₃ integrin (GenBank Accession Nos. U07375, L28832) andGP30 ligand (S68256) for erbB2.

[0151] Still other ligands include PDGF (GenBank Accession No. X03795,X02811), angiotensin (GenBank Accession No. K02215), and allRGD-containing peptides and proteins, such as ICAM-1 (GenBank AccessionNo. X06990) and VCAM-1 (GenBank Accession No. X53051) that bind tointegrin receptors. Other ligands include TNFα (GenBank Accession No.A21522, X01394), IFN-γ (GenBank Accession No. A11033, A11034), IGF-β1(GenBank Accession No. A29117, X56773, S61841, X56774, S61860), IGF-II(GenBank Accession No. A00738, X06159, Y00693), atrial naturieticpeptide (GenBank Accession No. X54669), endothelin-1 (GenBank AccessionNo. Y00749), coagulation factor Xa (GenBank Accession No. L00395,L00396, L29433, N00045, M14327), TGF-β1 (GenBank Accession No. A23751),IL-1α (GenBank Accession No. X03833), IL-1β (GenBank Accession No.M15330), and endoglin (GenBank Accession No. X72012).

[0152] a. Growth Factors

[0153] 1) Fibroblast Growth Factors

[0154] One family of growth factors that may be used within the contextof the present invention is the fibroblast growth factor (FGF) family.The members of the FGF family have a high degree of amino acid sequencesimilarities and common physical and biological properties, includingthe ability to bind to one or more FGF receptors.

[0155] This family of proteins includes FGFs designated FGF-1 (acidicFGF (aFGF)), FGF-2 (basic FGF (bFGF)), FGF-3 (int-2) (see, e.g., Mooreet al., EMBO J. 5:919-924, 1986), FGF-4 (hst-1/K-FGF) (see, e.g.,Sakamoto et al., Proc. Natl. Acad. Sci. USA 86:1836-1840, 1986; U.S.Pat. No. 5,126,323), FGF-5 (see, e.g., U.S. Pat. No. 5,155,217), FGF-6(hst-2) (see, e.g., published European Application EP 0 488 196 A2; Udaet al., Oncogene 7:303-309, 1992), FGF-7 (keratinocyte growth factor)(KGF) (see, e.g., Finch et al., Science 245:752-755, 1985; Rubin et al.,Proc. Natl. Acad. Sci. USA 86:802-806, 1989; and InternationalApplication WO 90/08771), FGF-8 (see, e.g., Tanaka et al., Proc Natl.Acad. Sci. USA 89:8528-8532, 1992); FGF-9 (see, Miyamoto et al., Mol.Cell. Biol. 13:4251-4259, 1993); FGF-11 (WO 96/39507); FGF-13 (WO96/39508); FGF-14 (WO 96/39506); FGF-15 (WO 96/39509). Otherpolypeptides that are reactive with an FGF receptor, that is anypolypeptide that specifically interacts with an FGF receptor, preferablythe high affinity FGF receptor, and is transported by way of endosomesinto the cell by virtue of its interaction with the FGF receptor aresuitable within the present invention.

[0156] DNA encoding FGF peptides and/or the amino acid sequences of FGFsare well known. For example, DNA encoding human FGF-1 (Jaye et al.,Science 233:541-545, 1986; U.S. Pat. No. 5,223,483), bovine FGF-2(Abraham et al., Science 233:545-548, 1986; Esch et al., Proc. Natl.Acad. Sci. USA 82:6507-6511, 1985; and U.S. Pat. No. 4,956,455), humanFGF-2 (Abraham et al., EMBO J. 5:2523-2528, 1986; U.S. Pat. No.4,994,559; U.S. Pat. No. 5,155,214; EP 470,183B; and Abraham et al.,Quant. Biol. 51:657-668, 1986) rat FGF-2 (see Shimasaki et al., Biochem.Biophys. Res. Comm., 1988, and Kurokawa et al., Nucleic Acids Res.16:5201, 1988), FGF-3, FGF-6, FGF-7 and FGF-9 are known (see also U.S.Pat. No. 5,155,214; U.S. Pat. No. 4,956,455; U.S. Pat. No. 5,026,839;U.S. Pat. No. 4,994,559, EP 0,488,196 A2, EMBL or GenBank databases, andreferences discussed herein).

[0157] FGFs exhibit a mitogenic effect on a wide variety of mesenchymal,endocrine and neural cells and are also important in differentiation anddevelopment. FGFs stimulate collateral vascularization and angiogenesis,which makes them useful as “payloads” as well, as discussed in asubsequent section. In some instances, FGF-induced mitogenic stimulationmay be detrimental. For example, cell proliferation and angiogenesis arean integral aspect of tumor growth. Members of the FGF family, includingFGF-2, are thought to play a pathophysiological role, for example, intumor development, rheumatoid arthritis, proliferative diabeticretinopathies and other complications of diabetes. To reduce oreliminate mitogenesis, muteins of FGF may be used and constructed asdescribed below. Such muteins retain the ability to bind to high and lowaffinity receptors.

[0158] Polypeptides reactive with FGF receptors are also useful intargeting not only tumors and malignant cells in particular, buthyperproliferating cells in general. Thus, to name one example, FGF-7,which is also known as KGF, can be used to target the vectors andconstructs of the present invention to hyperproliferating SMCs and avariety of epithelial cells. KGF is also particularly useful intargeting hepatocytes and type II pneumocytes of the lung.

[0159] The effects of FGFs are mediated by high affinity receptortyrosine kinases present on the cell surface of FGF-responsive cells(see, e.g., PCT WO 91/00916, WO 90/05522, PCT WO 92/12948; Imamura etal., Biochem. Biophys. Res. Comm. 155:583-590, 1988; Huang et al., J.Biol. Chem. 261:9568-9571, 1986; Partanen et al., EMBO J. 10:1347, 1991;and Moscatelli, J. Cell. Physiol. 131:123, 1987). Low affinity receptorsalso appear to play a role in mediating FGF activities. The highaffinity receptor proteins are single chain polypeptides with molecularweights ranging from 110 to 150 kD, depending on cell type thatconstitute a family of structurally related FGF receptors. Four FGFreceptor genes have been identified, three of which generate multiplemRNA transcripts via alternative splicing of the primary transcript.Some receptor specificity has been uncovered. For example, FGF-9 bindsspecifically to FGFR3, which is expressed in epithelial cells andcartilage rib bone, epithelial cells exclusively express FGFR3IIIb,while mesenchymal cells express FGFR3IIIb and FGFR3IIIc.

[0160] In addition to their use as ligands, various forms of FGFs may beused as “payloads” for gene therapy applications. While FGF cDNA orgenomic FGF DNA is often preferred for such therapeutic use, other formsmay efficaciously be used as well. The therapeutic aspects of FGF DNAuse are described in greater detail in Section F below.

[0161] 2) Vascular Endothelial Growth Factors

[0162] Vascular endothelial growth factors (VEGFs) can directlystimulate endothelial cell growth, enhance angiogenesis, enhance glucosetransport, and cause a rapid and reversible increase in blood vesselpermeability. VEGF is expressed during normal development and in certainnormal adult organs. Purified VEGF is a basic, heparin-binding,homodimeric glycoprotein that is heat-stable, acid-stable and may beinactivated by reducing agents. Polypeptides reactive with a VEGFreceptor are thus contemplated for use as ligands in the context of thepresent invention.

[0163] The members of this family have been referred to variously asvascular endothelial growth factor (VEGF), vascular permeability factor(VPF) and vasculotropin (see, e.g. Plouet et al., EMBO J. 8:3801-3806,1989). Herein, they are collectively referred to as VEGF.

[0164] DNA sequences encoding VEGF and methods to isolate thesesequences may be found primarily in U.S. Pat. No. 5,240,848, U.S. Pat.No. 5,332,671, U.S. Pat. No. 5,219,739, U.S. Pat. No. 5,194,596, andHouch et al., Mol. Endocrin. 5:180, 1991.

[0165] DNA encoding VEGF refers to DNA that encodes any such member ofthe VEGF family, including VEGF isoforms that result from alternativesplicing of RNA transcribed from a VEGF gene (see, e.g., InternationalPCT Application No. WO 90/13649, which is based on U.S. application Ser.Nos. 07/351,361, 07/369,424, 07/389,722, to GENENTECH, INC., and anyU.S. Patent based U.S. application Ser. Nos. 07/351,361, 07/369,424,07/389,722; European Patent Applications EP 0 506 477 A1 and EP 0 476983 A1 to Merck & Co.; Houck et al. (1991) Mol. Endo. 5:1806-1814). Itis also understood that substitutions in codons by virtue of thedegeneracy of the genetic code are encompassed by DNA encoding suchVEGF. DNA encoding the VEGF polypeptide may be obtained from any sourceknown to those of skill in the art; it may be isolated using standardcloning methods, synthesized or obtained from commercial sources,prepared as described in any of the above noted patents andpublications.

[0166] Four molecular species of VEGF result from alternative splicingof mRNA and contain 121, 165, 189 and 206 amino acids. The predominantisoform secreted by a variety of normal and transformed cells isVEGF₁₆₅. The secreted isoforms, VEGF₁₂₁, and VEGF₁₆₅ are preferred VEGFproteins. The longer isoforms, VEGF₁₈₉ and VEGF₂₀₆, bind to theextracellular matrix and need to be released by an agent, such assuramin, heparin or heparinase, or plasmin. VEGF₁₂₁, is a weakly acidicpolypeptide that lacks the heparin binding domain and, consequently,does not bind to heparin. Other preferred VEGF proteins contain variouscombinations of VEGF exons, such that the protein still binds VEGFreceptor and is internalized.

[0167] It is not necessary that a VEGF protein used as a ligand in thecontext of this invention either retain any of its in vivo biologicalactivities, such as stimulating endothelial cell growth, or bind heparinother than bind a VEGF receptor on a cell and be internalized. However,it may be desirable in certain contexts for VEGF to manifest certain ofits biological activities. For example, if VEGF is used as a carrier forDNA encoding a molecule useful in wound healing, it would be desirablethat VEGF exhibit vessel permeability activity and promotion offibroblast migration and angiogenesis. If VEGF is used as payload, asdescribed further in Section F below, retention of such abilities isalso desirable. It will be apparent from the teachings provided withinthe subject application which of the activities of VEGF are desirable tomaintain.

[0168] Quiescent and proliferating endothelial cells bind VEGF with highaffinity, and endothelial cell responses to VEGF appear to be mediatedby high affinity cell surface receptors (see, e.g., PCT Application WO92/14748, U.S. application Ser. No. 08/657,236, de Vries et al., Science255:9894-91, 1992; Terman et al., Biochem. Biophys. Res. Commun.187:1579-1586, 1992; Kendall et al., Proc. Natl. Acad. Sci. USA90:10705-10709, 1993; and Peters et al., Proc. Natl. Acad. Sci. USA90:8915-8919, 1993). Two tyrosine kinases have been identified as VEGFreceptors. The first, known as fms-like tyrosine kinase or FLT, is areceptor tyrosine kinase that is specific for VEGF. In adult andembryonic tissues, expression of FLT mRNA is localized to theendothelium and to populations of cells that give rise to endothelium.The second receptor, KDR (human kinase insert domain-containingreceptor), and its mouse homologue FLK-1, are closely related to FLT.The KDR/FLK-1 receptor is expressed in endothelium during the fetalgrowth stage, during earlier stages of embryonic development, and inadult tissues. In addition, messenger RNA encoding FLT and KDR have beenidentified in tumor blood vessels and specifically by endothelial cellsof blood vessels supplying glioblastomas. Similarly, FLT and KDR mRNAsare upregulated in tumor blood vessels in invasive human colonadenocarcinoma, but not in the blood vessels of adjacent normal tissues.

[0169] 3) Heparin-Binding Epidermal Growth Factors

[0170] HBEGF interacts with the same high affinity receptors as EGF onbovine aortic smooth muscle cells and human A431 epidermoid carcinomacells (Higashiyama, Science 251:936-939, 1991). HBEGFs exhibit amitogenic effect on a wide variety of cells including BALB/c 3T3fibroblast cells and smooth muscle cells, but are not mitogenic forendothelial cells (Higashiyama et al., Science 251:936-939, 1991).However, HBEGF has a stimulatory effect on collateral vascularizationand angiogenesis. Members of the HBEGF family are thought to play apathophysiological role, for example, in a variety of tumors, such asbladder carcinomas, breast tumors and non-small cell lung tumors. Thus,these cell types are likely candidates for delivery of therapeutic geneproducts.

[0171] HBEGF isolated from U-937 cells is heterogeneous in structure andcontains at least 86 amino acids and two sites of O-linked glycosylgroups (Higashiyama et al., J. Biol. Chem. 267:6205-6212, 1992). Thecarboxyl-terminal half of the secreted HBEGF shares approximately 35%sequence identity with human EGF, including six cysteines spaced in thepattern characteristic of members of the EGF protein family. Incontrast, the amino-terminal portion of the mature factor ischaracterized by stretches of hydrophilic residues and has no structuralequivalent in EGF. Site-directed mutagenesis of HBEGF and studies withpeptide fragments have indicated that the heparin-binding sequences ofHBEGF reside primarily in a 21 amino acid stretch upstream of andslightly overlapping the EGF-like domain.

[0172] DNA encoding an HBEGF peptide or polypeptide refers to any DNAfragment encoding an HBEGF, HBEGF fragment or HBEGF mutein that binds anEGF receptor and internalizes. Such DNA sequences encoding HBEGFfragments are available from publicly accessible databases, such as:EMBL, GenBank (Accession Nos. M93012 (monkey) and M60278 (human)); theplasmid pMTN-HBEGF (ATCC #40900) and pAX-HBEGF (ATCC #40899) (describedin PCT Application WO/92/06705); and Abraham et al., Biochem. Biophys.Res. Comm. 190:125-133, 1993).

[0173] The effects of HBEGFs are mediated by EGF receptor tyrosinekinases expressed on cell surfaces of HBEGF-responsive cells (see, e.g.,U.S. Pat. Nos. 5,183,884 and 5,218,090; and Ullrich et al., Nature309:4113-425, 1984). The EGF receptor proteins, which are single chainpolypeptides with molecular weights 170 kD, constitute a family ofstructurally related EGF receptors. Cells known to express the EGFreceptors include smooth muscle cells, fibroblasts, keratinocytes, andnumerous human cancer cell lines, such as the: A431 (epidermoid); KB3-1(epidermoid); COLO 205 (colon); CRL 1739 (gastric); HEP G2 (hepatoma);LNCAP (prostate); MCF-7 (breast); MDA-MB-468 (breast); NCI 417D (lung);MG63 (osteosarcoma); U-251 (glioblastoma); D-54 MB (glioma); and SW-13(adrenal).

[0174] For the purposes of this invention, if HBEGFs (includingfragments or derivatives thereof) are used as ligands, HBEGF need onlybind a specific EGF receptor and be internalized. Members of the HBEGFfamily are those that have sufficient nucleotide identity to hybridizeunder normal stringency conditions (typically greater than 75%nucleotide identity). Subfragments or subportions of a full-length HBEGFmay also be desirable. One skilled in the art may find from theteachings provided within that certain biological activities are more orless desirable, depending upon the application.

[0175] 2. Antibodies to Receptors that Internalize

[0176] Antibodies to molecules expressed on the surface of cells areuseful within the context of the present invention as long as theantibody is internalized following binding. Such antibodies include, butare not limited to, antibodies to FGF receptors, VEGF receptors,urokinase receptor, E- and P-selectins, VCAM-1, PDGF receptor, TGFreceptor, endosialin, alpha_(v) beta₃ integrin, LFA-1, E9 antigen, CD40,cadherins, and elk-1. Antibodies that are specific to cell surfacemolecules expressed by cells are readily generated as monoclonals orpolyclonal antisera. Many such antibodies are available (e.g., fromAmerican Type Culture Collection, Rockville, Md.). Alternatively,antibodies to ligands that bind/internalize may also be used. In such astrategy, the viral particles will have antibody on their surface, whichwill then be complexed to the ligand (see further discussion below).

[0177] Within the context of the present invention, antibodies areunderstood to include monoclonal antibodies, polyclonal antibodies,anti-idiotypic antibodies, antibody fragments (e.g., Fab, and F(ab′)₂,Fv variable regions, or complementarity determining regions). Antibodiesare generally accepted as specific against indolicidin analogues if theybind with a K_(d) of greater than or equal to 10⁻⁷M, preferably greaterthan of equal to 10⁻⁸M. The affinity of a monoclonal antibody or bindingpartner can be readily determined by one of ordinary skill in the art(see Scatchard, Ann. N.Y. Acad. Sci. 51:660-672, 1949). Once suitableantibodies have been obtained, they may be isolated or purified by manytechniques well known to those of ordinary skill in the art.

[0178] For example, one such internalizing, receptor-binding antibody isidentified herein as the “11A8” antibody. The 11A8 antibody is amonoclonal antibody which recognizes the high affinity fibroblast growthfactor receptor (FGFR1). The following general procedure, which may beused to generate other useful antibodies which bind to receptors andinternalize, was employed to generate 11A8.

[0179] Mice were immunized with intact SK-HEP1 cells, derived from aliver adenocarcinoma, which express high affinity FGF receptors.Hybridomas were screened using the extracellular domain of FGFR1. Oneresulting monoclonal, 11A8, recognizes the high affinity FGF receptor byWestern blotting, immunoprecipitates FGF receptor from cell extracts andrecognizes the native receptor on the cell surface. Immunofluorescencestudies show that 11A8 reacts with a membrane-associated protein on thesurface of SK-Hep-1 and SK-Mel-28 (human melanoma) cells, which alsoexpress numerous FGF receptors.

[0180] When 11A8 is conjugated to the ribosome inactivating proteinsaporin, it becomes a potent cytocidal agent that targets cells thatexpress high affinity FGF receptors. The resulting immunotoxin,11A8-saporin, inhibits solid tumor growth of a human melanomaxenografted into nude mice. When 11A8 is conjugated to nucleic acid orto the viral vectors of the present invention, it is able to targetcells expressing its cognate receptor and to deliver therapeutic genesequences to those cells, as it is able to internalize as well.

[0181] Commercially available antibodies to cell surface molecules mayalso be used as taught herein if they internalize. One assay that isused is a test for an antibody to kill cells. Briefly, the testhybridoma antibody and test cells are incubated. Unbound antibody iswashed away. A second stage antibody, such as an anti-IgG antibody,conjugated to saporin is incubated with the test cells. Cell killing isassessed by any known assay, including trypan blue exclusion, MTTuptake, fluorescein diacetate staining, and the like.

[0182] Other techniques may also be utilized to construct monoclonalantibodies (see Huse et al., Science 246:1275-1281, 1989; Sastry et al.,Proc. Natl. Acad. Sci. USA 86:5728-5732, 1989; Alting-Mees et al.,Strategies in Molecular Biology 3:1-9, 1990; describing recombinanttechniques). These techniques include cloning heavy and light chainimmunoglobulin cDNA in suitable vectors, such as λImmunoZap(H) andλImmunoZap(L). These recombinants may be screened individually orco-expressed to form Fab fragments or antibodies (see Huse et al.,supra; Sastry et al., supra). Positive plaques may subsequently beconverted to a non-lytic plasmid that allows high level expression ofmonoclonal antibody fragments from E. coli.

[0183] Similarly, portions or fragments, such as Fab and Fv fragments,of antibodies may also be constructed utilizing conventional enzymaticdigestion or recombinant DNA techniques to yield isolated variableregions of an antibody. Within one embodiment, the genes which encodethe variable region from a hybridoma producing a monoclonal antibody ofinterest are amplified using nucleotide primers for the variable region.

[0184] In addition, techniques may be utilized to change a “murine”antibody to a “human” antibody, without altering the binding specificityof the antibody. Some examples of the specific receptors against whichantibodies may be generated are set forth below.

[0185] a. Antibodies to Molecules on Tumor Cells

[0186] Antibodies to molecules expressed on the surface of tumor cellsare useful within the context of the present invention as long as theantibody is internalized following binding. Such antibodies include butare not limited to antibodies to FGF receptors, VEGF receptors, and thereceptors set forth above.

[0187] Antibodies may be polyclonal or monoclonal. Commerciallyavailable antibodies to some tumor cell surface molecules may be used ifthey internalize. One assay that is used is a test for an antibody tokill tumor cells. Briefly, the test hybridoma antibody and tumor cellsare incubated. Unbound antibody is washed away. A second stage antibody,such as an anti-IgG antibody, conjugated to saporin is incubated withthe tumor cells. Cell killing is assessed by any known assay, includingtrypan blue exclusion, MTT uptake, fluorescein diacetate staining, andthe like.

[0188] b. Antibodies to Molecules on Smooth Muscle Cells

[0189] Antibodies to molecules expressed on the surface of smooth musclecells are useful within the context of the present invention as long asthe antibody is internalized following binding. Such antibodies includebut are not limited to antibodies to FGF receptors, EGF receptors, TNFαreceptor, IFN-γ receptor, TGF receptor, endothelin 1 receptor.

[0190] Antibodies may be polyclonal or monoclonal. Commerciallyavailable antibodies to some smooth muscle cell surface molecules may beused if they internalize. Briefly, antibodies are raised by immunizationof mice, rats, rabbits or other animals with normal, tumorigenic, orcultured smooth muscle cells. Various immunization protocols may befound in for example, Harlow and Lane (Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988) and Coligan et al. (CurrentProtocols in Immunology, Greene Publishing, 1995). Followingimmunization, spleen or lymph nodes are collected for generatinghybridomas or serum is collected for polyclonal antibodies. Hybridomasare preferred. Cells from spleen or lymph node are fused to a myelomacell line (see, Harlow and Lane, supra; and Coligan et al., supra; forprotocols). Antibody-secreting hybridomas are grown, and the antibodiesare tested for binding to smooth muscle cells by ELISA, sectionstaining, flow cytometry, confocal microscopy and the like. When theantibodies are to be used on hyperproliferating smooth muscle cells,preferably the antibody does not bind or binds much less to quiescentsmooth muscle cells. Positive antibodies are further tested forinternalization. One assay that is used is a test for an antibody tokill smooth muscle cells. Briefly, the test hybridoma antibody andsmooth muscle cells are incubated. Unbound antibody is washed away. Asecond stage antibody, such as an anti-IgG antibody, conjugated tosaporin is incubated with the smooth muscle cells. Cell killing isassessed by any known assay, including trypan blue exclusion, MTTuptake, fluorescein diacetate staining, and the like.

[0191] C. Antibodies to Molecules on Endothelial and Smooth Muscle Cells

[0192] Antibodies to molecules expressed on the surface of endothelialand smooth muscle cells are useful within the context of the presentinvention as long as the antibody is internalized following binding.Such antibodies include but are not limited to antibodies to FGFreceptors, VEGF receptors, urokinase receptor, E- and P-selectins,VCAM-1, PDGF receptor, TGF receptor, endosialin, alpha_(v) beta₃integrin, LFA-1, E9 antigen, CD40, cadherins, and elk-1.

[0193] Antibodies may be polyclonal or monoclonal. Commerciallyavailable antibodies to some endothelial or smooth muscle cell surfacemolecules may be used if they internalize. One assay that is used is atest for an antibody to kill cells. Briefly, the test hybridoma antibodyand test cells are incubated. Unbound antibody is washed away. A secondstage antibody, such as an anti-IgG antibody, conjugated to saporin isincubated with the test cells. Cell killing is assessed by any knownassay, including trypan blue exclusion, MTT uptake, fluoresceindiacetate staining, and the like.

[0194] 3. Other Ligands

[0195] a. Ligands Internalized by Endothelial and Smooth Muscle Cells

[0196] As noted above, receptor-binding internalized ligands are used todeliver nucleic acids, including a therapeutic agent-encoding agent, toa cell expressing an appropriate receptor on its cell surface. Numerousmolecules that bind specific receptors have been identified and aresuitable for use in the present invention. In addition to ligands thattarget endothelial cells, ligands that target smooth muscle cells areuseful in the context of this invention. Smooth muscle cells (SMC) arean essential requirement for neovessel formation, providing thecontractile and structural components of capillaries, venules, veins,arterioles, and arteries. As such, targeting SMC will also affectneovascularization processes in diseased tissues. Such molecules includegrowth factors, cytokines, and antibodies. Many growth factors andfamilies of growth factors share structural and functional features andmay be used in the present invention. Families of growth factors includefibroblast growth factors FGF-1 through FGF-15, and vascular endothelialgrowth factor (VEGF). Other growth factors, such as PDGF(platelet-derived growth factor), TGF-α (transforming growth factor),TGF-β, HB-EGF, angiotensin and endoglin also bind to specific identifiedreceptors on cell surfaces and may be used in the present invention.Antibodies that are specific to cell surface molecules expressed byendothelial cells or smooth muscle cells are readily generated asmonoclonals or polyclonal antisera. Many such antibodies are available(e.g., from American Type Culture Collection, Rockville, Md.).Cytokines, including interleukins, CSFs (colony stimulating factors),and interferons, have specific receptors on endothelial cells, and maybe used as described herein. These and other ligands are discussed inmore detail below.

[0197] Fragments of these ligands may be used within the presentinvention, so long as the fragment retains the ability to bind to theappropriate cell surface molecule. Likewise, ligands with substitutionsor other alterations, but which retain binding ability, may also beused. As well, a particular ligand refers to a polypeptide(s) having anamino acid sequence of the native ligand, as well as modified sequences,(e.g., having amino acid substitutions, deletions, insertions oradditions compared to the native protein) as long as the ligand retainsthe ability to bind to its receptor on an endothelial cell and beinternalized.

[0198] b. Ligands that Bind to Tumor Cells

[0199] As noted above, receptor-binding internalized ligands are used todeliver nucleic acids to a cell expressing an appropriate receptor onits cell surface. Numerous molecules that bind specific receptors ontumor cells have been identified and are suitable for use in the presentinvention. For example, the following table sets forth some of thebetter known ligands and cell surface molecules on various tumors. TumorLigand Receptor T cell lymphomas IL-2 IL-2 receptor B cell lymphomasAntibody Immunoglobulin idiotypes Melanomas FGF MAGE; FGF receptorProstate tumors Prostate specific antigen-1; probasin Angiogenic tumorsFGF; VEGF; PDGF FGF receptor; VEGF receptor; PDGF receptor Breast tumorsheregulin; FGF erb B2; erb B3; erb B4; MUC-1; HSP-27; int-1; int-2Colon, lung tumors Antibody; FGF; CEA; FGF receptor; VEGF VEGF receptorBladder tumors HBEGF; EGF/TGF; EGF receptor; FGF receptor FGF Pancreatictumors FGF FGF receptor Myeloid leukemias FGF; CD antibodies FGFreceptor; CD molecules Endometrial VEGF VEGF receptor carcinoma;cervical carcinoma

[0200] In addition, other receptors, such as transferrin receptor, arepreferentially expressed on most all tumor cells. Antibodies that arespecific to cell surface molecules on tumors are readily generated asmonoclonals or polyclonal antisera. Many such antibodies are available(e.g., from American Type Culture Collection, Rockville, Md.).

[0201] Fragments of these ligands may be used within the presentinvention, so long as the fragment retains the ability to bind to theappropriate cell surface molecule. Likewise, ligands with substitutionsor other alterations, but which retain binding ability, may also beused. As well, a particular ligand refers to a polypeptide(s) having anamino acid sequence of the native ligand, as well as modified sequences,(e.g., having amino acid substitutions, deletions, insertions oradditions compared to the native protein) as long as the ligand retainsthe ability to bind to its receptor on a tumor cell and be internalized.

[0202] Some of the more useful receptors according to the presentinvention are those that efficiently bind ligand and not onlyinternalize it but enhance its delivery to the nucleus. Thus, ligandsthat specifically target such receptors are especially preferred ligandsthat specifically target receptors that direct the ligand to the nucleuswith high efficiency are even more preferred.

[0203] Ligands also encompass muteins that possess the ability to bindto their receptor expressing cells and be internalized. Such muteinsinclude, but are not limited to, those produced by replacing one or moreof the cysteines with serine as described herein. Typically, suchmuteins will have conservative amino acid changes. DNA encoding suchmuteins will, unless modified by replacement of degenerate codons,hybridize under conditions of at least low stringency to native DNAsequence encoding the wild-type ligand. (Exemplary methods of generatingFGF muteins are described in Example 3.)

[0204] DNA encoding a ligand may be prepared synthetically based onknown amino acid or DNA sequence, isolated using methods known to thoseof skill in the art (e.g., PCR amplification), or obtained fromcommercial or other sources. DNA encoding a ligand may differ from theabove sequences by substitution of degenerate codons or by encodingdifferent amino acids. Differences in amino acid sequences, such asthose occurring among the homologous ligand of different species as wellas among individual organisms or species, are tolerated as long as theligand binds to its receptor. Ligands may be isolated from naturalsources or made synthetically, such as by recombinant means or chemicalsynthesis.

[0205] Other receptor-binding ligands may be used in the presentinvention. Any protein, polypeptide, analogue, or fragment that binds toa cell-surface receptor and is internalized may be used. These ligandsmay be produced by recombinant or other means in preparation forconjugation to the nucleic acid binding domain. The DNA sequences andmethods to obtain the sequences of these receptor-binding internalizedligands are well known. For example, these ligands include CSF-1(GenBank Accession Nos. M11038, M37435; Kawasaki et al., Science230:291-296, 1985; Wong et al., Science 235:1504-1508, 1987); GM-CSF(GenBank Accession No. X03021; Miyatake et al., EMBO J. 4:2561-2568,1985); IFN-α (interferon) (GenBank Accession No. A02076; Patent No. WO8502862-A, Jul. 4, 1985); IFN-₇ (GenBank Accession No. A02137; PatentNo. WO 8502624-A, Jun. 20, 1985); IL-1-α (interleukin 1 alpha) (GenBankAccession No. X02531, M15329; March et al., Nature 315:641-647, 1985;Nishida et al., Biochem. Biophys. Res. Commun. 143:345-352, 1987);IL-1-P (interleukin 1 beta) (GenBank Accession No. X02532, M15330,M15840; March et al., Nature 315:641-647, 1985; Nishida et al., Biochem.Biophys. Res. Commun. 143:345-352, 1987; Bensi et al., Gene 52:95-101,1987); IL-1 (GenBank Accession No. K02770, M54933, M38756; Auron et al.,Proc. Natl. Acad. Sci. USA 81:7907-7911, 1984; Webb et al., Adv. GeneTechnol. 22:339-340, 1985); IL-2 (GenBank Accession No. A14844, A21785,X00695, X00200, X00201, X00202; Lupker et al., Patent No. EP 0307285-A,Mar. 15, 1989; Perez et al., Patent No. EP 0416673-A, Mar. 13, 1991;Holbrook et al., Nucleic Acids Res. 12:5005-5013, 1984; Degrave et al.,EMBO J. 2:2349-2353, 1983; Taniguchi et al., Nature 302:305-310, 1983);IL-3 (GenBank Accession No. M14743, M20137; Yang et al., Cell 47:3-10,1986; Otsuka et al., J. Immunol. 140:2288-2295, 1988); IL-4 (GenBankAccession No. M13982; Yokota et al., Proc. Natl. Acad. Sci. USA83:5894-5898, 1986); IL-5 (GenBank Accession No. X04688, J03478; Azumaet al., Nucleic Acids Res. 14:9149-9158, 1986; Tanabe et al., J. Biol.Chem. 262:16580-16584, 1987); IL-6 (GenBank Accession No. Y00081,X04602, M54894, M38669, M14584; Yasukawa et al., EMBO J. 6:2939-2945,1987; Hirano et al., Nature 324:73-76, 1986; Wong et al., Behring Inst.Mitt. 83:40-47, 1988; May et al., Proc. Natl. Acad. Sci. USA83:8957-8961, 1986); IL-7 (GenBank Accession No. J04156; Goodwin et al.,Proc. Natl. Acad. Sci. USA 86:302-306, 1989); IL-8 (GenBank AccessionNo. ZI 1686; Kusner et al., Kidney Int. 39:1240-1248, 1991); IL-10(GenBank Accession No. X78437, M57627; Vieira et al., Proc. Natl. Acad.Sci. USA 88:1172-1176, 1991); IL-1I (GenBank Accession No. M57765M37006; Paul et al., Proc. Natl. Acad. Sci. USA 87:7512-7516, 1990);IL-13 (GenBank Accession No. X69079, U10307; Minty et al., Nature362:248-250, 1993; Smirnov, Shemyakin and Ovchinnikov Institute ofBioorganic Chemistry, Jun. 2, 1994); TNF-α (Tumor necrosis factor)(GenBank Accession No. A21522; Patent No. GB 2246569-A1, Feb. 5, 1992);TNF-β (GenBank Accession No. D12614; Matsuyama et al., FEBS LETTERS302:141-144, 1992); urokinase/urokinase receptor (GenBank Accession Nos.X02760/X74309); α-1,3 fucosyl transferase, α1-antitrypsin/E-selectin(GenBank Accession Nos. M98825, D38257/M87862); P-selectin glycoproteinligand, P-selectin ligand/P-selectin (GenBank Accession Nos. U25955,U02297/L01574); VCAM11VLA-4 integrin receptor (GenBank Accession Nos.X53051/X16983 and L12002); E9 (Blann et al., Atherosclerosis 120:221,1996)/TGFβ receptor; Fibronectin (GenBank Accession No. X02761);typeI^(α1) collagen (GenBank Accession No. Z74615), type I β2-collagen(GenBank Accession No. Z74616), hyaluronic acid/CD44 (GenBank AccessionNo. M59040); CD40 ligand (GenBank Accession No. L07414)/CD40 (GenBankAccession No. M83312); EFL-3, LERTK-2 ligands (GenBank Accession Nos.L37361, U09304) for elk-1 (GenBank Accession No. M25269); VE-cadherin(GenBank Accession No. X79981) ligand for catenins; ICAM-3 (GenBankAccession No. X69819) ligand for LFA-1, and von Willebrand Factor(GenBank Accession No. X04385), fibrinogen and fibronectin (GenBankAccession No. X92461 ligands for α_(v)β₃ integrin (GenBank AccessionNos. U07375, L28832) and GP30 ligand (S68256) for erbB2. DNA sequencesof other suitable receptor-binding internalized ligands may be obtainedfrom GenBank or EMBL DNA databases, reverse-synthesized from proteinsequence obtained from PIR database or isolated by standard methods(Sambrook et al., supra) from cDNA or genomic libraries.

[0206] c. Other Ligands That Bind to Cells

[0207] Other receptor-binding ligands may be used in the presentinvention. Any protein, polypeptide, analogue, or fragment that binds toa smooth muscle cell-surface receptor and is internalized may be used.These ligands may be produced by recombinant or other means inpreparation for conjugation to the nucleic acid binding domain. The DNAsequences and methods to obtain the sequences of these receptor-bindinginternalized ligands are well known. For example, these ligands includePDGF (GenBank Accession No. X03795, X02811), angiotensin (GenBankAccession No. K02215), and all RGD-containing peptides and proteins,such as ICAM-1 (GenBank Accession No. X06990) and VCAM-1 (GenBankAccession No. X53051) that bind to integrin receptors. Other ligandsinclude TNFα (GenBank Accession No. A21522, X01394), IFN-γ (GenBankAccession No. A11033, Al 1034), IGF-I (GenBank Accession No. A29117,X56773, S61841, X56774, S61860), IGF-II (GenBank Accession No. A00738,X06159, Y00693), atrial naturietic peptide (GenBank Accession No.X54669), endothelin-1 (GenBank Accession No. Y00749), coagulation factorXa (GenBank Accession No. L00395, L00396, L29433, N00045, M14327),TGF-β1 (GenBank Accession No. A23751), IL-1α (GenBank Accession No.X03833), IL-1α (GenBank Accession No. M15330), and endoglin (GenBankAccession No. X72012). DNA sequences of other suitable receptor-bindinginternalized ligands may be obtained from GenBank or EMBL DNA databases,reverse-synthesized from protein sequence obtained from PIR database orisolated by standard methods (Sambrook et al., supra) from cDNA orgenomic libraries.

[0208] As noted previously, any protein, polypeptide, analogue, orfragment that binds to a cell-surface receptor and is internalized maybe used. Molecules that mimic or interact with a cell surface moleculethat is trafficked to the nucleus are also included within the scope ofthe present invention. These ligands may be produced by recombinant orother means in preparation for conjugation to the nucleic acid bindingdomain. The DNA sequences and methods to obtain the sequences of thesereceptor-binding internalized ligands are well known. For example, theseligands and ligand/receptor pairs include urokinase/urokinase receptor(GenBank Accession Nos. X02760/X74309); α-1,3 fucosyl transferase,α1-antitrypsin/E-selectin (GenBank Accession Nos. M98825,D38257/M87862); P-selectin glycoprotein ligand, P-selectinligand/P-selectin (GenBank Accession Nos. U25955, U02297/L23088),VCAM1/VLA-4 (GenBank Accession Nos. X53051/X16983); E9 antigen (Blann etal., Atherosclerosis 120:221, 1996)/TGFβ receptor; Fibronectin (GenBankAccession No. X02761); type I α1-collagen (GenBank Accession No.Z74615), type I β2-collagen (GenBank Accession No. Z74616), hyaluronicacid/CD44 (GenBank Accession No. M59040); CD40 ligand (GenBank AccessionNo. L07414)/CD40 (GenBank Accession No. M83312); ELF-3, LERTK-2 ligands(GenBank Accession Nos. L37361, U09304) for elk-1 (GenBank Accession No.M25269); VE-cadherin (GenBank Accession No. X79981); ligand forcatenins; ICAM-3 (GenBank Accession No. X69819) ligand for LFA-1, andvon Willebrand Factor (GenBank Accession No. X04385), fibrinogen andfibronectin (GenBank Accession No. X92461) ligands for α_(v)β₃ integrin(GenBank Accession Nos. U07375, L28832). DNA sequences of other suitablereceptor-binding internalized ligands may be obtained from GenBank orEMBL DNA databases, reverse-synthesized from protein sequence obtainedfrom PIR database or isolated by standard methods (Sambrook et al.,supra) from cDNA or genomic libraries.

[0209] d. Peptidomimetic Ligands

[0210] Ligands or fragments thereof that bind to a cell-surface receptorand are internalized, but which are mimetics of “true” polypeptides, arealso contemplated for use in the present invention. Thus, in one aspect,the invention contemplates the preparation and use of non-peptidepeptidomimetics useful for mimicking the activity of peptides, whichmakes peptidomimetics additional sources of targeting ligands that maybe attached to the viral vectors of the present invention.

[0211] Methods of generating and identifying peptidomimetics useful asdescribed herein are known in the art; (see, e.g., WO 93/17032). Forexample, the aforementioned application describes a process of preparingpeptidomimetic compounds useful for mimicking the activity of peptidesand described the peptide-like activity of one such mimetic. Similarly,the production of peptidomimetic drugs via utilizing chemically modifiedmoieties to mimic antibody structure, based on conformation studies, isdescribed in U.S. Pat. No. 5,331,573. Methods of testing the drugs soprepared is also disclosed therein. Peptidomimetics of antibodies arethus useful as disclosed herein, not only as ligands but as moleculesuseful in linking viral particles to targeting ligands.

[0212] Other useful peptidomimetic molecules useful as ligands and/or“linkers” herein are described in published International App. No. WO9220704; Brandt, et al., Antimicrob Agents Chemother, 40:1078, 1996;Sepp-Lorenzino, et al., Cancer Res, 55:5302, 1995; and Chander et al., JPharm Sci, 84:404, 1995. Notwithstanding the fact that such mimetics arenot true peptides, various covalent and non-covalent means of linkingsuch peptidomimetic molecules to viral proteins may be used as disclosedherein.

[0213] e. Selection of Ligands that Bind Other Cell Surface Molecules

[0214] Ligands for use in the present invention may also be selected bya method such as phage display (see, for example, U.S. Pat. No.5,223,409.) Briefly, in this method, DNA sequences are inserted into thegene III or gene VIII gene of a filamentous phage, such as M13. Severalvectors with multicloning sites have been developed for insertion(McLafferty et al., Gene 128:29-36, 1993; Scott and Smith, Science249:386-390, 1990; Smith and Scott, Methods Enzymol. 217:228-257, 1993).Using tumor cell targeting as an example, the inserted DNA sequences maybe randomly generated or be variants of a known binding domain forbinding tumor cells. Single chain antibodies may readily be generatedusing this method. Generally, the inserts encode from 6 to 20 aminoacids. The peptide encoded by the inserted sequence is displayed on thesurface of the bacteriophage. Bacteriophage expressing a binding domainfor tumor cells are selected for by binding to tumor cells. Unboundphage are removed by a wash, typically containing 10 mM Tris, 1 mM EDTA,and without salt or with a low salt concentration. Bound phage areeluted with a salt containing buffer, for example. The NaClconcentration is increased in a step-wise fashion until all the phageare eluted. Typically, phage binding with higher affinity will bereleased by higher salt concentrations.

[0215] Eluted phage are propagated in the bacteria host. Further roundsof selection may be performed to select for a few phage binding withhigh affinity. The DNA sequence of the insert in the binding phage isthen determined. Once the predicted amino acid sequence of the bindingpeptide is known, sufficient peptide for use herein as an nucleic acidbinding domain may be made either by recombinant means or synthetically.Recombinant means is used when the receptor-binding internalizedligand/nucleic acid binding domain is produced as a fusion protein. Thepeptide may be generated as a tandem array of two or more peptides, inorder to maximize affinity or binding.

[0216] f. Identifying and Isolating Internalizing Molecules

[0217] Any and all molecules that mimic or interact with a cell surfacemolecule that is trafficked to the nucleus are also included within thescope of the present invention. One exemplary method of identifying andisolating such molecules is essentially as follows.

[0218] First, one may identify a cell or tissue of interest—e.g., a cellto which one wishes to target a therapeutic moiety. Next, generateantibodies—preferably, monoclonal antibodies—to the cell surface of theputative target cell. Identify and isolate hybridomas secreting theantibodies to the cell surface. Methods of generating monoclonalantibodies and of identifying hybridomas producing said antibodies areknown in the art.

[0219] As a next step, the monoclonal antibodies are admixed with aculture of target cells and allowed to incubate for a predeterminedperiod of time. Subsequently, a second antibody is added to theadmixture—that is, an antibody to the first antibody (anti-idiotypeantibody). Preferably, the second antibody is toxic to the target cellupon internalization and delivery to the nucleus; in this manner, celldeath is indicative of internalization of the first antibody, to whichthe second antibody is bound.

[0220] By screening the cell cultures, one may readily identify andisolate all killing antibody complexes and may separate out theantibodies that possess the ability to translocate to the nucleus. Suchantibodies may then be used according to the methods disclosedherein—e.g., they may be used to target and deliver viral vectors totarget cell populations.

[0221] Although the foregoing example discusses the generation andidentification of antibodies, it is understood that useful ligands ofthe present invention are not restricted to antibodies. Any molecule,for example, that mimics the ability of FGF and/or FGFR to be traffickeddirectly to the nucleus is contemplated for use as disclosed herein.Thus, any molecule that interacts with a cell surface molecule that istrafficked to the nucleus is contemplated by the present disclosure.

[0222] 2. Modification of Receptor-Binding Internalized Ligands

[0223] The ligands for use herein may be customized for a particularapplication. Means for modifying proteins is provided below. Briefly,additions, substitutions and deletions of amino acids may be produced byany commonly employed recombinant DNA method. Modified peptides,especially those lacking proliferative function, and chimeric peptides,which retain their specific binding and internalizing activities, arealso contemplated for use herein. Modifications also include theaddition or deletion of residues, such as the addition of a cysteine tofacilitate conjugation and to form conjugates that contain a definedmolar ratio (e.g., 1:1) of the polypeptides (see, e.g., U.S. Pat. No.5,175,147; PCT Application No. WO 89/00198, U.S. Ser. No. 07/070,797;PCT Application No. WO 91/15229; and U.S. Ser. No. 07/505,124). Stillother useful modifications include adding sequence that are subject topost-translational modification (e.g., myristylation, palmatylation,phophorylation, ribosylation) that improve or alter protein function,stability or the like.

[0224] As noted above, any ligand that binds to a cell surface receptorand is internalized may be used within the context of this invention.Such ligands may be polypeptides or peptide analogues, includingpeptidomimetics. Ligands also include fragments thereof, or constrainedanalogues of such peptides that bind to the receptor and internalize alinked targeted agent. Members of the FGF family, including FGF-1 toFGF-15, are preferred. Modified peptides, especially those lackingproliferative function, and chimeric peptides, which retain the specificbinding and internalizing activities are also contemplated for useherein.

[0225] Modification of the polypeptide may be effected by any meansknown to those of skill in this art. The preferred methods herein relyon modification of DNA encoding the polypeptide and expression of themodified DNA. DNA encoding one of the receptor-binding internalizedligands discussed above may be mutagenized using standard methodologies.For example, cysteine residues that are responsible for aggregateformation may be deleted or replaced. If necessary, the identity ofcysteine residues that contribute to aggregate formation may bedetermined empirically, by deleting and/or replacing a cysteine residueand ascertaining whether the resulting protein aggregates in solutionscontaining physiologically acceptable buffers and salts. In addition,fragments of these receptor-binding internalized ligands may beconstructed and used. The binding region of many of these ligands havebeen delineated. For example, the receptor binding region of FGF2 hasbeen identified by mutation analysis and FGF peptideagonists/antagonists to reside between residues 33-77 and between102-129 of the 155 amino acid form (Baird et al., PNAS 85:2324; Ericksonet al., Biochem. 88:3441). Exons 1-4 of VEGF are required for receptorbinding. Fragments may also be shown to bind and internalize by any oneof the tests described herein.

[0226] Mutations may be made by any method known to those of skill inthe art, including site-specific or site-directed mutagenesis of DNAencoding the protein and the use of DNA amplification methods usingprimers to introduce and amplify alterations in the DNA template, suchas PCR splicing by overlap extension (SOE). Site-directed mutagenesis istypically effected using a phage vector that has single- anddouble-stranded forms, such as M13 phage vectors, which are well-knownand commercially available. Other suitable vectors that contain asingle-stranded phage origin of replication may be used (see, e.g.,Veira et al., Meth. Enzymol. 15:3, 1987). In general, site-directedmutagenesis is performed by preparing a single-stranded vector thatencodes the protein of interest (i.e., a member of the FGF family or atherapeutic molecule, such as an intrabody). An oligonucleotide primerthat contains the desired mutation within a region of homology to theDNA in the single-stranded vector is annealed to the vector followed byaddition of a DNA polymerase, such as E. coli DNA polymerase I (Klenowfragment), which uses the double stranded region as a primer to producea heteroduplex in which one strand encodes the altered sequence and theother the original sequence. The heteroduplex is introduced intoappropriate bacterial cells and clones that include the desired mutationare selected. The resulting altered DNA molecules may be expressedrecombinantly in appropriate host cells to produce the modified protein.

[0227] Suitable conservative substitutions of amino acids are well-knownand may be made generally without altering the biological activity ofthe resulting molecule. For example, such substitutions are generallymade by interchanging within the groups of polar residues, chargedresidues, hydrophobic residues, small residues, and the like. Ifnecessary, such substitutions may be determined empirically merely bytesting the resulting modified protein for the ability to bind to andinternalize upon binding to the appropriate receptors. Those that retainthis ability are suitable for use in the conjugates and methods herein.As such, an amino acid residue of a receptor-binding internalized ligandis non-essential if the polypeptide that has been modified by deletionor alteration of the residue possesses substantially the same ability tobind to its receptor and internalize a linked agent as the unmodifiedpolypeptide.

[0228] As used herein, “biological activity” generally refers to theactivity of a compound or a physiological response that results upon invivo administration of a compound, composition or other mixture.Biological activity thus encompasses therapeutic effects andpharmaceutical activity of such compounds, compositions, complexes, andmixtures. Biological activity also refers to the ability of a moleculeto bind to a cell, to internalize and to localize to the nucleus.Biological activity may be determined with reference to particular invitro activities as measured in a defined assay. For example, within thecontext of this invention, a biological activity of FGF, or fragments ofFGF, is the ability of FGF to bind to cells bearing FGF receptors andinternalize a linked agent. This activity may be assessed in vitro,e.g., by conjugating FGF to a cytotoxic agent (such as saporin),contacting cells bearing FGF receptors (e.g., fibroblasts) with theconjugate, and assessing cell proliferation or inhibition of growth. Invivo activity may be determined using recognized animal models, such asthe mouse xenograft model for anti-tumor activity (see, e.g., Beitz etal., Cancer Research 52:227-230, 1992; Houghton et al., Cancer Res.42:535-539, 1982; Bogden et al., Cancer (Philadelphia) 48:10-20, 1981;Hoogenhout et al., Int. J. Radiat. Oncol., Biol. Phys. 9:871-879, 1983;Stastny et al., Cancer Res. 53:5740-5744, 1993).

[0229] Binding to a receptor followed by internalization are the onlyactivities required for a ligand to be suitable for use herein. However,some of the ligands are growth factors and cause mitogenesis. Forexample, all of the FGF proteins induce mitogenic activity in a widevariety of normal diploid mesoderm-derived and neural crest-derivedcells. A test of such “FGF mitogenic activity,” which reflects theability to bind to FGF receptors and to be internalized, is the abilityto stimulate proliferation of cultured bovine aortic endothelial cells(see, e.g., Gospodarowicz et al., J. Biol. Chem. 257:12266-12278, 1982;Gospodarowicz et al., Proc. Natl. Acad. Sci. USA 73:4120-4124, 1976).Muteins with reduced mitogenic activity are made by the methodsdescribed herein. In the Examples, FGF muteins with reduced mitogenicactivity have been constructed by site-directed mutagenesis. Non- orreduced-mitogenic proteins can also be constructed by swapping thereceptor-binding domain with the receptor-binding domain of a relatedprotein. By way of example, the domain of FGF2 may be swapped with thereceptor-binding domain of FGF7 to create an FGF that does not causeproliferation and may alter the binding profile.

[0230] If the FGF or other ligand has been modified so as to lackmitogenic activity or other biological activities, binding andinternalization may still be readily assayed by any one of the followingtests or other equivalent tests. Generally, these tests involve labelingthe ligand, incubating it with target cells, and visualizing ormeasuring intracellular label. For example, briefly, FGF may befluorescently labeled with FITC or radiolabeled with ¹²⁵I.Fluorescein-conjugated FGF is incubated with cells and examinedmicroscopically by fluorescence microscopy or confocal microscopy forinternalization. When FGF is labeled with ¹²⁵I, the labeled FGF isincubated with cells at 4° C. Cells are temperature shifted to 37° C.and washed with 2 M NaCl at low pH to remove any cell-bound FGF. Labelis then counted and thereby measuring internalization of FGF.

[0231] Alternatively, in another method of assaying the binding andinternalization abilities of a ligand, the ligand can be conjugated witha nucleic acid binding domain by any of the methods described herein andcomplexed with a plasmid encoding saporin or conjugated with saporin orother cytotoxic molecule and assessed for cytotoxicity. As discussedbelow, the complex may be used to transfect cells and cytotoxicitymeasured.

[0232] Finally, muteins of the FGFs are known to those of skill in theart (see, e.g., U.S. Pat. No. 5,175,147; PCT Application No. WO89/00198, U.S. Ser. No. 07/070,797; PCT Application No. WO 91/15229; andU.S. Ser. No. 07/505,124). Such muteins may also be used according tothe teachings of the present invention.

[0233] F. Payload

[0234] 1. Therapeutic-Product-Encoding Molecules

[0235] Molecules that encode therapeutic products, which are alsoreferred to herein as therapeutic nucleic acids, are molecules thateffect a treatment upon or within a cell, generally by modifying genetranscription of translation. Therapeutic nucleic acids of the presentinvention may be used in the context of “positive” or “negative” genetherapy, depending on the effect one seeks to achieve.

[0236] For example, a therapeutic nucleotide sequence may encode all ora portion of a gene. If it encodes all (or the most critical functionalportions) of a gene, it may effect genetic therapy by serving as areplacement for a defective gene. Such a sequence may also function byrecombining with DNA already present in a cell, thereby replacing adefective portion of a gene.

[0237] A variety of positive gene therapy applications and therapeuticgene products are described hereinbelow and include such diverseapplications as the treatment of ischemia, the promotion of woundhealing, the stimulation of bone growth and regrowth, increasedangiogenesis, and the like. The replacement of a defective ornonfunctional gene with one that produces the desired gene product isalso considered “positive” gene therapy, whether one is replacing adysfunctional or nonfunctional regulatory sequence or a sequence thatencodes a structural protein.

[0238] Similarly, “negative” gene therapy is encompassed by the presentinvention as well. Thus, therapeutic nucleic acids of the presentinvention may encode products that reduce or halt hyperproliferativediseases (e.g. of SMCs; restenosis is one example), tumor formation andgrowth, metastasis, and the like, to name a few examples.

[0239] Further details regarding both positive and negative gene therapyapplications are set forth below in subsequent sections of thespecification. The following illustrations are thus intended to beexemplary and not limiting.

[0240] a. Gene Products for the Treatment of Ischemia

[0241] For example, in ischemia, endothelial and smooth muscle cellsfail to proliferate. A construct that expresses FGF, alone or incombination with FGF protein to give short-term relief and induce FGFreceptor, can be used to combat effects of ischemia. In such a case, FGFgene with a leader sequence to promote secretion is preferable. As well,the FGF gene is preferably driven by a constitutive promoter. Inaddition, muteins of the FGFs are known to those of skill in the art andmay be useful as payload molecules as well as ligands. (See, e.g., U.S.Pat. No. 5,175,147; PCT Application No. WO 89/00198, U.S. Ser. No.07/070,797; PCT Application No. WO 91/15229; and U.S. Ser. No.07/505,124.)

[0242] Other useful sequences which may be delivered using the vectorsof the present invention include those encoding human superoxidedismutase (SOD) and analogs thereof (see, e.g., U.S. Pat. No. 5,455,029,5,130,245 and 4,742,004) as well as opiod peptides (see, e.g., U.S. Pat.No. 4,684,624). Other sequences which encode therapeutic products usefulas disclosed herein, whose GenBank numbers are provided in SectionF.1.e. below, include sequences encoding IGF (see, e.g., U.S. Pat. Nos.5,612,198 and 5,324,639); TGFβ1, TGFβ2, and TGFβ3 (see, e.g., U.S. Pat.Nos. 5,168,051, 5,482,851, 4,886,747, and 5,221,620); hepatocyte growthfactor (HGF) (see, e.g., U.S. Pat. Nos. 5,547,856; 5,328,837; and5,316,921); PDGF A (see, e.g., U.S. Pat. Nos. 5,605,816 and 5,219,759);and PDGF B (see, e.g., U.S. Pat. Nos. 5,272,064, 5,665,567).

[0243] Nucleic acid sequences encoding the following therapeuticproducts are also useful as payloads according to the present invention:VEGF 121, VEGF 165, FGF1, FGF2, FGF4, and FGF5. Sequence information forthese molecules is provided elsewhere herein.

[0244] Finally, in all instances in which reference is made topublications, particularly patent applications and patents, it shouldgenerally be understood that the disclosures of all patent documentsrecited herein are incorporated by reference, as though fully set forthherein.

[0245] In addition, individuals afflicted with certain angiogenicdiseases suffer from a paucity of angiogenic factor and may thus bedeficient in microvasculature. Certain aspects of reproduction, such asovulation, repair of the uterus after menstruation, and placentaldevelopment depend on angiogenesis. For reproductive disorders withunderlying angiogenic dysfunction, a construct that expresses FGF, VEGF,or other angiogenic factors may be beneficial. Useful sequences encodingsuch angiogenic factors are described in various sections herein,including sections E. 1.a. and E.1.b.

[0246] b. Oligonucleotides

[0247] The conjugates provided herein may also be used to deliver aribozyme, deoxyribozyme, antisense oligonucleotide, and the like totargeted cells. These nucleic acids may be present in the complex ofligand and nucleic acid binding domain or encoded by a nucleic acid inthe complex. Alternatively, the nucleic acid may be directly linked tothe ligand. Such products include antisense RNA, antisense DNA,ribozymes, triplex-forming oligonucleotides, and oligonucleotides thatbind proteins. The nucleic acids can also include RNA traffickingsignals, such as viral packaging sequences (see, e.g., Sullenger et al.(1994) Science 262:1566-1569).

[0248] Nucleic acids and oligonucleotides for use as described hereincan be synthesized by any method known to those of skill in this art(see, e.g., WO 93/01286, U.S. application Ser. No. 07/723,454; U.S. Pat.No. 5,218,088; U.S. Pat. No. 5,175,269; U.S. Pat. No. 5,109,124).Identification of oligonucleotides and ribozymes for use as antisenseagents and DNA encoding genes for targeted delivery for genetic therapyinvolve methods well known in the art. For example, the desirableproperties, lengths and other characteristics of such oligonucleotidesare well known. Antisense oligonucleotides are typically designed toresist degradation by endogenous nucleolytic enzymes by using suchlinkages as: phosphorothioate, methylphosphonate, sulfone, sulfate,ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and othersuch linkages (see, e.g., Agrwal et al., Tetrahedron Lett. 28:3539-3542(1987); Miller et al., J. Am. Chem. Soc. 93:6657-6665 (1971); Stec etal., Tetrahedron Lett. 26:2191-2194 (1985); Moody et al., Nucl. AcidsRes. 12:4769-4782 (1989); Uznanski et al., Nucl. Acids Res. (1989);Letsinger et al., Tetrahedron 40:137-143 (1984); Eckstein, Annu. Rev.Biochem. 54:367-402 (1985); Eckstein, Trends Biol. Sci. 14:97-100(1989); Stein In: Oligodeoxynucleotides. Antisense Inhibitors of GeneExpression, Cohen, Ed, Macmillan Press, London, pp. 97-117 (1989); Jageret al., Biochemistry 27:7237-7246 (1988)).

[0249] Antisense nucleotides are oligonucleotides that bind in asequence-specific manner to nucleic acids, such as mRNA or DNA. Whenbound to mRNA that has complementary sequences, antisense preventstranslation of the mRNA (see, e.g., U.S. Pat. No. 5,168,053 to Altman etal.; U.S. Pat. No. 5,190,931 to Inouye, U.S. Pat. No. 5,135,917 toBurch; U.S. Pat. No. 5,087,617 to Smith and Clusel et al. (1993) Nucl.Acids Res. 21:3405-3411, which describes dumbbell antisenseoligonucleotides). Triplex molecules refer to single DNA strands thatbind duplex DNA forming a colinear triplex molecule, thereby preventingtranscription (see, e.g., U.S. Pat. No. 5,176,996 to Hogan et al., whichdescribes methods for making synthetic oligonucleotides that bind totarget sites on duplex DNA).

[0250] Particularly useful antisense nucleotides and triplex moleculesare molecules that are complementary or bind to the sense strand of DNAor mRNA that encodes a protein involved in cell proliferation, such asan oncogene or growth factor, (e.g., bFGF, int-2, hst-1/K-FGF, FGF-5,hst-2/FGF-6, FGF-8). Other useful antisense oligonucleotides includethose that are specific for IL-8 (see, e.g., U.S. Pat. No. 5,241,049),c-src, c-fos H-ras (lung cancer), K-ras (breast cancer), urokinase(melanoma), BCL2 (T-cell lymphoma), IGF-1 (glioblastoma), IGF-1 receptor(glioblastoma), TGF-β1, and CRIPTO EGF receptor (colon cancer). Theseparticular antisense plasmids reduce tumorigenicity in athymic andsyngeneic mice.

[0251] These nucleic acids or nucleic acids that encode antisense can belinked to bFGF for the treatment of psoriasis. Anti-senseoligonucleotides or nucleic acids encoding antisense specific fornonmuscle myosin heavy chain and/or c-myb (see, e.g., Simons et al.(1992) Circ. Res. 70:835-843; PCT Application WO 93/01286, U.S.application Ser. No. 07/723,454: LeClerc et al. (1991) J. Am. Coll.Cardiol. 17 (2 Suppl. A):105A; Ebbecke et al. (1992) Basic Res. Cardiol.87:585-591) can be targeted by an FGF, for example to inhibit smoothmuscle cell proliferation, such as occurs following angioplasty.

[0252] A ribozyme is an RNA molecule that specifically cleaves RNAsubstrates, such as mRNA, resulting in inhibition or interference withcell growth or expression. There are at least five known classes ofribozymes involved in the cleavage and/or ligation of RNA chains.Ribozymes can be targeted to any RNA transcript and can catalyticallycleave such transcript (see, e.g., U.S. Pat. No. 5,272,262; U.S. Pat.No. 5,144,019; and U.S. Pat. Nos. 5,168,053, 5,180,818, 5,116,742 and5,093,246 to Cech et al.). Any such ribozyme or nucleic acid encodingthe ribozyme may be delivered to a cell bearing a receptor for areceptor-internalized binding ligand.

[0253] Ribozymes and the like may be delivered to the targeted cells byDNA encoding the ribozyme linked to a eukaryotic promoter, such as aneukaryotic viral promoter, such that upon introduction into the nucleus,the ribozyme will be directly transcribed. In such instances, theconstruct will also include a nuclear translocation sequence, generallyas part of the ligand or as part of a linker between the ligand andnucleic acid binding domain.

[0254] c. Prodrugs

[0255] A nucleic acid molecule encoding a prodrug may alternatively beused within the context of the present invention. Prodrugs are inactivein the host cell until either a substrate or an activating molecule isprovided. Most typically, a prodrug activates a compound with little orno cytotoxicity into a toxic compound. Three of the more often usedprodrug molecules, all of which are suitable for use in the presentinvention, are nitroreductase, thymidine kinase (e.g. HSVtk) andcytosine deaminase (e.g. E. coli CD).

[0256] Briefly, a wide variety of gene products which either directly orindirectly activate a compound with little or no cytotoxicity into atoxic product may be utilized within the context of the presentinvention. Representative examples of such gene products include HSVTK(herpes simplex virus thymidine kinase) and VZVTK (Varicella zostervirus thymidine kinase), which selectively phosphorylate certain purinearabinosides and substituted pyrimidine compounds. Phosphorylationconverts these substrates (compounds) to metabolites that are cytotoxicor cytostatic. For example, exposure of the drugs ganciclovir,acyclovir, or any of their analogues (e.g., FIAU, FIAC, DHPG) to cellsexpressing HSVTK allows conversion of the drug into its correspondingactive nucleotide triphosphate form.

[0257] Other gene products that may be utilized within the context ofthe present invention include E. coli guanine phosphoribosyltransferase, which converts thioxanthine into toxic thioxanthinemonophosphate (Besnard et al., Mol. Cell. Biol. 7:4139-4141, 1987);alkaline phosphatase, which converts inactive phosphorylated compoundssuch as mitomycin phosphate and doxorubicin-phosphate to toxicdephosphorylated compounds; fungal (e.g., Fusarium oxysporum) orbacterial cytosine deaminase, which converts 5-fluorocytosine to thetoxic compound 5-fluorouracil (Mullen, PNAS 89:33, 1992);carboxypeptidase G2, which cleaves glutamic acid from para-N-bis(2-chloroethyl) aminobenzoyl glutamic acid, thereby creating a toxicbenzoic acid mustard; and Penicillin-V amidase, which convertsphenoxyacetabide derivatives of doxorubicin and melphalan to toxiccompounds (see generally, Vrudhula et al., J. Med. Chem. 36:919-923,1993; Kern et al., Canc. Immun. Immunother. 31:202-206, 1990). Moreover,a wide variety of Herpesviridae thymidine kinases, including bothprimate and non-primate herpesviruses, are suitable. Such herpesvirusesinclude Herpes Simplex Virus Type 1 (McKnight et al., Nuc. Acids Res8:5949-5964, 1980), Herpes Simplex Virus Type 2 (Swain and Galloway, J.Virol. 46:1045-1050, 1983), Varicella Zoster virus (Davison and Scott,J. Gen. Virol. 67:1759-1816, 1986), marmoset herpesvirus (Otsuka andKit, Virology 135:316-330, 1984), feline herpesvirus type 1 (Nunberg etal., J. Virol. 63:3240-3249, 1989), pseudorabies virus (Kit and Kit,U.S. Pat. No. 4,514,497, 1985), equine herpesvirus type 1 (Robertson andWhalley, Nuc. Acids Res. 16:11303-11317, 1988), bovine herpesvirus type1 (Mittal and Field, J. Virol 70:2901-2918, 1989), turkey herpesvirus(Martin et al., J. Virol. 63:2847-2852, 1989), Marek's disease virus(Scott et al., J. Gen. Virol. 70:3055-3065, 1989), herpesvirus saimiri(Honess et al., J. Gen. Virol. 70:3003-3013, 1989) and Epstein-Barrvirus (Baer et al., Nature (London) 310:207-311, 1984). Suchherpesviruses may be readily obtained from commercial sources such asthe American Type Culture Collection (“ATCC”, Rockville, Md.).

[0258] Furthermore, as indicated above, a wide variety of inactiveprecursors may be converted into active inhibitors. For example,thymidine kinase can phosphorylate nucleosides (e.g., dT) and nucleosideanalogues such as ganciclovir (9-{[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl} guanosine), famciclovir, buciclovir, penciclovir,valciclovir, acyclovir (9-[2-hydroxy ethoxy)methyl] guanosine),trifluorothymidine, 1-[2-deoxy, 2-fluoro, beta-D-arabinofuranosyl]-5-iodouracil, ara-A (adenosine arabinoside, vivarabine),1-beta-D-arabinofuranoxyl thymine, 5-ethyl-2′-deoxyuridine,5-iodo-5′-amino-2,5′-dideoxyuridine, idoxuridine(5-iodo-2′-deoxyuridine), AZT (3′ azido-3′ thymidine), ddC(dideoxycytidine), AIU (5-iodo-5′ amino 2′,5′-dideoxyuridine) and AraC(cytidine arabinoside).

[0259] Other gene products may render a cell susceptible to toxicagents. Such products include tumor necrosis factor, viral proteins, andchannel proteins that transport drugs.

[0260] A cytocide-encoding agent may be constructed as a prodrug, whichwhen expressed in the proper cell type is processed or modified to anactive form. For example, the saporin gene may be constructed with an N-or C-terminal extension containing a protease-sensitive site. Theextension renders the protein inactive and subsequent cleavage in a cellexpressing the appropriate protease restores enzymatic activity.

[0261] d. Tumor Suppressor Genes

[0262] The definition of a tumor suppressor gene has recently beenbroadened to include genes (and their products) which are subject tofrequent downregulation in cancer, suggestive of an importanttumor-suppressing activity despite the lack of mutation. Examples of theforegoing include cell adhesion molecules such as E-cadherin (GenBankAccession No. Z 18923), which play a role in tissue development andepithelial cell differentiation. E-cadherin expression correlates withepithelial differentiation, whereas loss of E-cadherin expressionpromotes epithelial dedifferentiation and invasiveness of humancarcinoma cells. Thus, the restoration of E-cadherin function preventsinvasiveness of epithelial tumor cells.

[0263] Human BGP (biliary glycoprotein) also mediates cell adhesion in amanner similar to the cadherins. Thus, BGP (GenBank Accession No.J03858) is another gene which may be used within the context of thepresent invention.

[0264] Other tumor-suppressor genes useful according to the presentinvention include the following. It should be noted, however, that thislist is not exhaustive, only exemplary: Rb (GenBank Accession No.M15400); p53 (GenBank Accession Nos. X02469, M60950); CDKN2/P16/MTS1(GenBank Accession No. S78535,U12818); PTEN/MMAC1 (GenBank Accession No.U92436); APC (GenBank Accession No. M74088); p331NG1 (GenBank AccessionNo. AF001954); Smad4 (GenBank Accession No. U59914); maspin (GenBankAccession No. U04313); von Hippel-Lindau (VHL) (GenBank Accession Nos.AF010238, U19763, U68055,U687176,U49746); Wilms tumor (WTI) (GenBankAccession No. X69950); Bin1 (GenBank Accession No. U68485); Men1(GenBank Accession Nos. U93237, U93326); Neurofibromatosis 2 (NF2)(GenBank Accession No. L27065); MXI1 (GenBank Accession No. L07648): andFHIT (GenBank Accession No. U46922).

[0265] e. Vascularization and Tissue Repair

[0266] A wide variety of therapeutic nucleic acid sequences encodingtherapeutic gene products involved in vascularization, wound healing(e.g. the healing of chronic ulcers) and tissue repair, including therepair of connective tissue (e.g. bone), are appropriate for use inconjunction with the constructs, vectors and methods of the presentinvention. Sequences encoding the following VEGF and VEGF-relatedproteins and polypeptides are particularly useful for such applicationsand include the following: VEGF (Bovine; GenBank Accession No. M32976);VEGF (Bovine; GenBank Accession No. M31836); VEGF-C (GenBank AccessionNo. X94216); VEGF-B (GenBank Accession No. U48801); VEGF (GenBankAccession No. X62568); Angiopoietin-1 (GenBank Accession No. U83508);Angiogenin (GenBank Accession No. M11567); IGF-1 (GenBank Accession No.X03563); IGF-II (GenBank Accession Nos. X03562, M13970, M14116, M14117,M14118); HGF (GenBank Accession Nos. X16323, S80567); PDGF A (GenBankAccession No. X03795); PDGF B (GenBank Accession Nos. X02744, X02811);TGFBI (GenBank Accession No. A23751); TGFB2 (GenBank Accession No.A23752); and TGFB3 (GenBank Accession No. A23753).

[0267] Still other useful therapeutic nucleotide sequences encode thefollowing molecules—many of which are particularly useful in the repairof connective tissue, such as bone: PTH (GenBank Accession Nos. J00301,V00597); BMP1 (GenBank Accession Nos. M22488, Y08723; see also U.S. Pat.No. 5,108,922); BMP2(GenBank Accession No. M22489; also see U.S. Pat.No. 5,013,649); BMP3 (GenBank Accession No. M22491; see also U.S. Pat.No. 5,116,738); BMP5 (see U.S. Pat. Nos. 5,635,373 and 5,106,748); BMP6(see U.S. Pat. No. 5,187,076); BMP7 (see U.S. Pat. No. 5,141,905); BMP8(see U.S. Pat. No. 5,688,678); BMP10 (see U.S. Pat. No. 5,637,480);BMP11 (see U.S. Pat. No. 5,639,638); mammalian BMPs (see U.S. Pat. No.5,620,867); tissue differentiation affecting factor (see U.S. Pat. No.5,679,783); morphogenic protein OP-3 (see U.S. Pat. No. 5,652,118);osteoinductive factors (see U.S. Pat. No. 4,877,864); osteogenicproteins (see U.S. Pat. Nos. 5,106,626, 4,968,590, and RE35694); andXenopus BMPs (see U.S. Pat. No. 5,670,338).

[0268] The foregoing represent but a few examples of useful therapeuticsequences and gene products that may be utilized in tissue repair andrevascularization. As noted below, many of those same sequences areuseful in the repair of connective tissues, such as bone, and othertissue injuries.

[0269] For bone repair, sequences encoding bone morphogenic proteins(BMPs), parathyroid hormone (PTH) and insulin-like growth factors (IGFs)are of particular usefulness. The following genes are thus appropriatefor use as payloads according to the teachings of the present invention:PTH (GenBank Accession Nos. J00301, V00597); BMP1 (GenBank AccessionNos. M22488, Y08723); BMP2 (GenBank Accession No. M22489); BMP3 (GenBankAccession No. M22491); IGF-1 (GenBank Accession No. X03563); and IGF-II(GenBank Accession Nos. X03562, M13970, 71. M14116, M14117, M14118).Sequences encoding other BMPs such as BMP4, BMP5, BMP6, and the like arealso useful as disclosed herein.

[0270] f. Apoptosis-Inducing Agents

[0271] There are many agents, both of a chemical and proteinaceousnature, that can induce apoptosis. Therefore, apoptosis-inducing agentsare also therapeutic agents within the context of the present invention.Examples of nucleotide sequences which encode such agents, whichsequences may be delivered with high specificity using the vectors ofthe present intention, include the following. As before, this listing isexemplary and is neither exhaustive nor limiting of the invention: p53(GenBank Accession Nos. X02469, M60950); c-myc (GenBank Accession No.E01841); TNF-alpha (GenBank Accession No. E02870); Fas ligand (GenBankAccession No. U08137; see also U.S. Pat. Nos. 5,663,070 and 5,652,210;p38-mitogen activated protein (MAP) kinase (GenBank Accession No.L35253); and IFN-gamma (GenBank Accession Nos. E06017, A11033).

[0272] g. Cytocidal Gene Products

[0273] A cytocide-encoding agent is a nucleic acid molecule (e.g., DNAor RNA) that, upon internalization by a cell, and subsequenttranscription (if DNA) and[/or] translation into a cytocidal agent, iscytotoxic or cytostatic, to a cell, for example, by inhibiting cellgrowth through interference with protein synthesis or through disruptionof the cell cycle.

[0274] Cytocides include saporin, the ricins, abrin, gelonin, otherribosome inactivating proteins, Pseudomonas exotoxin, diphtheria toxin,angiogenin, tritin, dianthins 32 and 30, momordin, pokeweed antiviralprotein, mirabilis antiviral protein, bryodin, angiogenin, and shigaexotoxin, as well as other cytocides that are known to those of skill inthe art. Inhibitors of cell cycle are well known.

[0275] DNA molecules that encode an enzyme that results in cell death orrenders a cell susceptible to cell death upon the addition of anotherproduct are preferred. For example, saporin is an enzyme that cleavesrRNA and inhibits protein synthesis. Other enzymes that inhibit proteinsynthesis are especially well suited for use in the present invention.Alternatively, the product may be a ribozyme, antisense, or othernucleic acid molecule that causes cell death.

[0276] Ribosome-inactivating proteins (RIPs), which include ricin,abrin, and saporin, are plant proteins that catalytically inactivateeukaryotic ribosomes. Ribosome-inactivating proteins inactivateribosomes by interfering with the protein elongation step of proteinsynthesis. For example, the ribosome-inactivating protein saporin (alsoreferred to as SAP) has been shown to inactivate 60S ribosomes bycleavage of the N-glycosidic bond of the adenine at position 4324 in therat 28S ribosomal RNA (rRNA).

[0277] Several structurally related ribosome inactivating proteins havebeen isolated from seeds and leaves of the plant Saponaria officinalis(soapwort) (GB Patent 2;194,241 B; GP Patent 2,216,891; EP Patent89306016). Saporin proteins for use in this invention have amino acidsequences found in the natural plant host Saponaria officinalis (e.g.,SEQ ID NO. 22) or modified sequences, such as amino acid substitutions,deletions, insertions or additions, but that still express substantialribosome inactivating activity. Several molecular isoforms of theprotein are also known. Any of these saporin proteins or modifiedproteins that are cytotoxic may be used in the present invention. Othersuitable saporin polypeptides include other members of the multi-genefamily coding for isoforms of saporin-type ribosome inactivatingproteins including SO-1 and SO-3 (Fordham-Skelton et al., Mol. Gen.Genet. 221:134-138, 1990), SO-2 (see, e.g., U.S. application Ser. No.07/885,242; GB 2,216,891; see also Fordham-Skelton et al., Mol. Gen.Genet. 229:460-466, 1991), S0-4 (see, e.g., GB 2,194,241 B; see alsoLappi et al., Biochem. Biophys. Res. Commun. 129:934-942, 1985) and S0-5(see, e.g., GB 2,194,241 B; see also Montecucchi et al., Int. J. PeptideProtein Res. 33:263-267, 1989). Any such protein, or portion thereof,that exhibits cytotoxicity in standard in vitro or in vivo assays withinat least about an order of magnitude of the saporin conjugates describedherein is contemplated for use herein.

[0278] Ribosome inactivating protein encoding DNA sequences may usemammalian-preferred codons (SEQ. ID NO. 23). Preferred codon usage asexemplified in Current Protocols in Molecular Biology, infra, and Zhanget al. (Gene 105:61, 1991) for mammals, yeast, Drosophila, E. coli, andprimates is established for saporin sequences.

[0279] In addition to saporin discussed above, other cytocides thatinhibit protein synthesis are useful in the present invention. The genesequences for these cytocides may be isolated by standard methods, suchas PCR, probe hybridization of genomic or cDNA libraries, antibodyscreenings of expression libraries, or clones may be obtained fromcommercial or other sources. The DNA sequences of many of thesecytocides are well known, including ricin A chain (GenBank Accession No.X02388); maize ribosome inactivating protein (GenBank Accession No.L26305); gelonin (GenBank Accession No. L12243; PCT Application WO92/03155; U.S. Pat. No. 5,376,546; diphtheria toxin (GenBank AccessionNo. K01722); trichosanthin (GenBank Accession No. M34858); tritin(GenBank Accession No. D13795); pokeweed antiviral protein (GenBankAccession No. X78628); mirabilis antiviral protein (GenBank AccessionNo. D90347); dianthin 30 (GenBank Accession No. X59260); abrin (GenBankAccession No. X55667); shiga (GenBank Accession No. M19437) andPseudomonas exotoxin (GenBank Accession Nos. K01397, M23348). When DNAsequences or amino acid sequences are known, DNA molecules encodingthese proteins may be synthesized, and may contain mammalian-preferredcodons.

[0280] The therapeutic product-encoding agent, such as saporin DNAsequence, is introduced into a plasmid in operative linkage with anappropriate promoter for expression of polypeptides in the organism. Theplasmid can optionally include sequences, such as origins of replicationthat allow for the extrachromosomal maintenance of thesaporin-containing plasmid, or can be designed to integrate into thegenome of the host (as an alternative means to ensure stable maintenancein the host).

[0281] In addition to saporin discussed above, other cytocides thatinhibit protein synthesis are useful in the present invention. The genesequences for these cytocides may be isolated by standard methods, suchas PCR, probe hybridization of genomic or cDNA libraries, antibodyscreenings of expression libraries, or clones may be obtained fromcommercial or other sources. The DNA sequences of many of thesecytocides are well known, including ricin A chain (GenBank Accession No.X02388); maize ribosome inactivating protein (GenBank Accession No.L26305); gelonin (GenBank Accession No. L12243; PCT Application WO92/03155; U.S. Pat. No. 5,376,546; diphtheria toxin (GenBank AccessionNo. K01722); trichosanthin (GenBank Accession No. M34858); tritin(GenBank Accession No. D13795); pokeweed antiviral protein (GenBankAccession No. X78628); mirabilis antiviral protein (GenBank AccessionNo. D90347); dianthin 30 (GenBank Accession No. X59260); abrin (GenBankAccession No. X55667); shiga (GenBank Accession No. M19437) andPseudomonas exotoxin (GenBank Accession Nos. K01397, M23348). When DNAsequences or amino acid sequences are known, DNA molecules encodingthese proteins may be synthesized, and may contain mammalian-preferredcodons.

[0282] 2. Promoters and Additional Elements

[0283] A therapeutic product-encoding agent of the present invention,such as a DNA sequence, is generally introduced into a plasmid inoperative linkage with an appropriate promoter for expression ofpolypeptides in the recipient cells. The plasmid can optionally includesequences such as origins of replication that allow for theextrachromosomal maintenance of the saporin-containing plasmid, or canbe designed to integrate into the genome of the host (as an alternativemeans to ensure stable maintenance in the host).

[0284] In general, constructs will also contain elements necessary fortranscription and translation. The choice of the promoter will dependupon the cell type to be transformed and the degree or type of controldesired. Promoters can be constitutive or active in any cell type,tissue specific, cell specific, event specific, temporal-specific orinducible. Cell-type specific promoters and event type specificpromoters are preferred. Examples of constitutive or nonspecificpromoters include the SV40 early promoter (U.S. Pat. No. 5,118,627), theSV40 late promoter (U.S. Pat. No. 5,118,627), CMV early gene promoter(U.S. Pat. No. 5,168,062), and adenovirus promoter. In addition to viralpromoters, cellular promoters are also amenable within the context ofthis invention. In particular, cellular promoters for the so-calledhousekeeping genes are useful. Viral promoters are preferred, becausegenerally they are stronger promoters than cellular promoters.

[0285] Tissue specific promoters are particularly useful for expressionin a wide variety of cells, including endothelial and smooth musclecells. By using one of this class of promoters, an extra margin ofspecificity can be attained. SMC-specific promoters are particularlyuseful in targeting proliferative diseases involving SMC. For example,FGFR promoter, EGFR promoter, PDGF receptor promoter, integrin receptorpromoters, α-actin promoter, SM1 and SM2 myosin heavy chain promoters,calponin-h1 promoter, SM22 alpha angiotensin receptor promoter, areuseful within the context of this invention.

[0286] Exemplary tissue-specific promoters include alpha-crystalline,tyrosinase, α-fetoprotein, prostate specific antigen, CEA, α-actin, VEGFreceptor, erbB-2, C-myc, cyclin D, FGF receptor, gamma-crystallinepromoter, tek, tie, urokinase receptor, E-selectin, P-selectin, VCAM-1,endoglin, endosialin, alpha_(v) integrin, β₃ integrin, endothelin-1,ICAM-3, E9, von Willebrand Factor, CD-44, CD40, vascular endothelialcadherin, notch 4 and high molecular weight melanoma-associated antigen.

[0287] Endothelial-specific promoters are especially useful in targetingproliferative diseases involving endothelial cells. For treatingdiseases dependent or exacerbated by angiogenesis or primary angiogenicdiseases, the following promoters are especially useful: VEGF-receptorpromoter (Morishita et al., J. Biol. Chem. 270:27948, 1995; GenBankAccession No. X89776); FGF receptor promoter; TEK or tie 2 promoter, areceptor tyrosine kinase expressed predominately in endothelium ofactively growing blood vessels (Huang et al., Oncogene 11:2097, 1995;GenBank Accession No. L06139); tie (WO 96/09381; Korhonen et al., Blood86:1828, 1995; GenBank Accession No. X60954; GenBank Accession No.S89716); urokinase receptor, which is expressed at high levels inendothelial cells during angiogenesis (Hollas et al., Cancer Res.51:3690, 1991; Gum et al., Anti-Cancer Res. 15:1167, 1995; Soravia etal., Blood 86:624, 1995; GenBank Accession No. S78532); E- andP-selectin, which has increased expression in endothelium of tumors,such as breast (Fox et al., J. Pathol 177:369, 1995; Biancone et al., J.Exp. Med. 183:581, 1996; GenBank Accession No. M64485; GenBank AccessionNo. L01874); VCAM-1 (Iademarco et al., J. Biol. Chem. 267:16323, 1992;GenBank Accession No. M92431); endoglin, which is upregulated in thevasculature of tumors (Bellon et al., Eur. J. Immunol. 23:2340, 1993;Gougos and Letarte, J. Biol. Chem. 265:8361, 1990; GenBank Accession No.HSENDOG); endosialin, expressed preferentially in tumor capillaries(Rettig et al., PNAS 89:10832, 1992); alpha V-beta3 integrin(Villa-Garcia et al., Blood 3:668, 1994; Donahue et al., BBA 1219:228,1994); endothelin-1, a growth factor for endothelial cells (GenBankAccession No. M25377; GenBank Accession No. J04819; GenBank AccessionNo. J05489); ICAM-3, expressed in tumor endothelium (Patey et al., Am.J. Pathol. 148:465, 1996; Fox et al., J Path. 177:369, 1995; GenBankAccession No. S50015); E9 antigen, upregulated in tumor endothelium(Wang et al., Int. J. Cancer 54:363, 1993); von Willebrand factor(Jahroudi and Lynch, Mol. Cell. Biol. 14:999, 1994; GenBank AccessionNo. HUMVWFI; GenBank Accession No. HUMVWFA); CD44 (Hofmann et al.,Cancer Res. 53:1516, 1993; Maltzman et al., Mol. Cell. Biol. 16:2283,1996; GenBank Accession No. HUMCD44B); CD40 (Pammer et al., Am. J.Pathol. 148:1387, 1996; GenBank Accession No. HACD40L; GenBank AccessionNo. HSCD405FR); vascular-endothelial cadherin, highly expressed inendothelial cells of hemangiomas (Martin-Padura et al., J. Pathol.175:51, 1995); notch 4 (Uyttendaele et al., Development 122:2251, 1996)and high molecular weight melanoma-associated antigen.

[0288] Inducible promoters may also be used. These promoters includeMMTV LTR (PCT WO 91/13160), inducible by dexamethasone, metallothionein,inducible by heavy metals, and promoters with cAMP response elements,inducible by cAMP. By using an inducible promoter, the nucleic acid maybe delivered to a cell and will remain quiescent until the addition ofthe inducer. This allows further control on the timing of production ofthe gene product.

[0289] Event-type specific promoters are active or up-regulated onlyupon the occurrence of an event, such as tumorigenicity or viralinfection. The HIV LTR is a well known example of an event-specificpromoter. The promoter is inactive unless the tat gene product ispresent, which occurs upon viral infection. Some event-type promotersare also tissue-specific.

[0290] Additionally, promoters that are coordinately regulated with aparticular cellular gene may be used. For example, promoters of genesthat are coordinately expressed when a particular FGF receptor gene isexpressed may be used. Then, the nucleic acid will be transcribed whenthe FGF receptor, such as FGFR1, is expressed, and not when FGFR2 isexpressed. This type of promoter is especially useful when one knows thepattern of FGF receptor expression in a particular tissue, so thatspecific cells within that tissue may be killed upon transcription of acytotoxic agent gene without affecting the surrounding tissues.

[0291] If the domain binds in a sequence specific manner, the constructmust contain the sequence that binds to the nucleic acid binding domain.As described below, the target nucleotide sequence may be containedwithin the coding region of the cytocide, in which case, no additionalsequence need be incorporated. Additionally, it may be desirable to havemultiple copies of target sequence. If the target sequence is codingsequence, the additional copies must be located in non-coding regions ofthe cytocide-encoding agent. The target sequences of the nucleic acidbinding domains are typically generally known. If unknown, the targetsequence may be readily determined. Techniques are generally availablefor establishing the target sequence (e.g., see PCT Application WO92/05285 and U.S. Serial No. 586,769).

[0292] In addition to the promoter, repressor sequences, negativeregulators, or tissue-specific silencers may be inserted to reducenon-specific expression of the cytocide or prodrug. Multiple repressorelements may be inserted in the promoter region. Repression oftranscription is independent on the orientation of repressor elements ordistance from the promoter. One type of repressor sequence is aninsulator sequence. Such sequences inhibit transcription (Dunaway etal., Mol Cell Biol 17: 182-9, 1997; Gdula et al., Proc Natl Acad Sci USA93:9378-83, 1996, Chan et al., J Virol 70: 5312-28, 1996; Scott andGeyer, EMBO J. 14: 6258-67, 1995; Kalos and Fournier, Mol Cell Biol 15:198-207, 1995; Chung et al., Cell 74: 505-14, 1993) and will silencebackground transcription.

[0293] Negative regulatory elements have been characterized in thepromoter regions of a number of different genes. The repressor elementfunctions as a repressor of transcription in the absence of factors,such as steroids, as does the NSE in the promoter region of theovalbumin gene (Haecker et al., Mol. Endocrinology 9:1113-1126, 1995).These negative regulatory elements bind specific protein complexes fromoviduct, none of which are sensitive to steroids. Three differentelements are located in the promoter of the ovalbumin gene.Oligonucleotides corresponding to portions of these elements repressviral transcription of the TK reporter. One of the silencer elementsshares sequence identity with silencers in other genes (TCTCTCCNA).

[0294] Repressor elements have also been identified in the promoterregion of collagen II gene. Gel retardation studies showed that nuclearfactors from HeLa cells bind specifically to DNA fragments containingthe silencer region, whereas chondrocyte nuclear extracts did not showany binding activity (Savanger et al., J. Biol. Chem. 265(12):6669-6674,1990). Repressor elements have also been shown to regulate transcriptionin the carbamyl phosphate synthetase gene (Goping et al., Nucleic AcidResearch 23(10):1717-1721, 1995). This gene is expressed in only twodifferent cell types, hepatocytes and epithelial cells of the intestinalmucosa. Negative regulatory regions have also been identified in thepromoter region of the choline acetyltransferase gene, the albuminpromoter (Hu et al., J. Cell Growth Differ. 3(9):577-588, 1992),phosphoglycerate kinase (PGK-2) gene promoter (Misuno et al., Gene119(2):293-297, 1992), and in the6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene, in which thenegative regulatory element inhibits transcription in non-hepatic celllines (Lemaigre et al., Mol. Cell Biol. 11(2):1099-1106). Furthermore,the negative regulatory element Tse-1 has been identified in a number ofliver specific genes, including tyrosine aminotransferase (TAT). TATgene expression is liver specific and inducible by both glucocorticoidsand the cAMP signaling pathway. The cAMP response element (CRE) has beenshown to be the target for repression by Tse-1 and hepatocyte-specificelements (Boshart et al., Cell 61(5):905-916, 1990).

[0295] In preferred embodiments, elements that increase the expressionof the desired product are incorporated into the construct. Suchelements include internal ribosome binding sites (IRES; Wang andSiddiqui, Curr. Top. Microbiol. Immunol 203:99, 1995; Ehrenfeld andSemler, Curr. Top. Microbiol. Immunol. 203:65, 1995; Rees, et al.,Biotechniques 20:102, 1996; Sugimoto et al., Biotechnology 12:694,1994). IRES increase translation efficiency. As well, other sequencesmay enhance expression. For some genes, sequences especially at the 5′end inhibit transcription and/or translation. These sequences areusually palindromes that can form hairpin structures. Any such sequencesin the nucleic acid to be delivered are generally deleted. Expressionlevels of the transcript or translated product are assayed to confirm orascertain which sequences affect expression. Transcript levels may beassayed by any known method, including Northern blot hybridization,Rnase probe protection and the like. Protein levels may be assayed byany known method, including ELISA.

[0296] Other elements may be incorporated into the construct. Inpreferred embodiments, the construct includes a transcription terminatorsequence, including a polyadenylation sequence, splice donor andacceptor sites, and an enhancer. Other elements useful for expressionand maintenance of the construct in mammalian cells or other eukaryoticcells may also be incorporated (e.g., origin of replication). Becausethe constructs are conveniently produced in bacterial cells, elementsthat are necessary or enhance propagation in bacteria are incorporated.Such elements include an origin of replication, selectable marker andthe like (see discussion below).

[0297] An additional level of control for initiating expression of thenucleic acid only in appropriate cells or enhancing uptake of complex isthe delivery of two constructs, one of which encodes the cytocide andthe other construct encodes a second gene that controls expression ofthe promoter driving the cytocide or prodrug or enhances uptake of thecomplexes into tumor masses or other target cells. By way of example, onone construct, the cytocide encoding agent is controlled by a promoter,such as a heat shock promoter. The second construct is a gene, such as agene that elicits SOS pathway under control of a tumor-specificpromoter. The two constructs are co-delivered or sequentially delivered.When delivered into tumor cells, the SOS gene is expressed and resultsin causing expression of the cytocide-encoding agent. In this case, thetwo constructs could be merged into one construct.

[0298] In the other type of multiple delivery system, the firstconstruct is a cytocide gene under control of a promoter, such as thosedescribed above. The second construct comprises a different promotercontrolling expression of a gene, such as IL-2, that induces leakinessin a tumor mass to allow better penetration. When the second constructis introduced first, the tumor mass will be more readily accessible forthe first construct to be delivered.

[0299] Typically, the constructs are plasmid vectors. A preferredconstruct is a modified pNASS vector (Clontech, Palo Alto, Calif.). Inthe modified vector, amp. R gene is replaced by kan. R gene, a poly Asignal sequence is added upstream of the mammalian promoter. A T7promoter is added downstream of the mammalian promoter and upstream ofthe cytocide or prodrug gene to facilitate verification of cytotoxicactivity. Other suitable mammalian expression vectors are well known(see, e.g., Ausubel et al., 1995; Sambrook et al., supra; Invitrogencatalogue, San Diego, Calif.; Novagen, Madison, Wis.; Pharmaciacatalogue, Uppsala, Sweden; and others).

[0300] G. Formulation and Administration of Pharmaceutical Compositions

[0301] The retargeted viral vectors and complexes provided herein areuseful in the treatment and prevention of various diseases. Whilecertain diseases are listed below as examples, it is to be understoodthat the vectors, complexes, conjugates, and other constructs disclosedherein are useful in a wide variety of therapeutic applications,including the treatment of proliferative disease, quiescent disease, andmetabolic disease. As noted previously, the origin of the disease isirrelevant; thus, whether the condition or disease is genetic,congenital, or acquired, the compositions and methods of the presentinvention are particularly useful in therapeutic interventions.

[0302] As used herein, “treatment” or “therapy” means any manner inwhich the symptoms of a condition, disorder or disease are amelioratedor otherwise beneficially altered. Treatment also encompasses anypharmaceutical use of the compositions herein, whether said uses are invivo, ex vivo, or in vitro. As used herein, “amelioration” of thesymptoms of a particular disorder refers to any lessening, whetherpermanent or temporary, lasting or transient, that can be attributed toor associated with administration of the composition.

[0303] 1. Treatment of Tumors

[0304] As noted above, the compositions of the present invention areused to treat tumors. In these diseases, cell growth is excessive oruncontrolled. Tumors suitable for treatment within the context of thisinvention include, but are not limited to, breast tumors, gliomas,melanomas, prostate cancer, hepatomas, sarcomas, lymphomas, leukemias,ovarian tumors, thymomas, nephromas, pancreatic cancer, colon cancer,head and neck cancer, stomach cancer, lung cancer, mesotheliomas,myeloma, neuroblastoma, retinoblastoma, cervical cancer, uterine cancer,and squamous cell carcinoma of skin. As discussed above, ligands forthese cancers bind to cell surface receptors that are generallypreferentially expressed in tumors. Many of these cell surface receptorsand their ligands are known. For tumors without such ligand-receptorpairs, ligands, such as antibodies, can be developed.

[0305] Through delivery of the compositions of the present invention,unwanted growth of cells may be slowed or halted, thus ameliorating thedisease. The methods utilized herein specifically target and kill orhalt proliferation of tumor cells having receptors for the ligand ontheir surfaces. This treatment is suitable for warm-blooded animals:mammals, including, but not limited to, humans, horses, dogs, and cats,and for non-mammals, such as avian species. Methods of treating suchanimals with these FGF conjugates are provided herein. These conjugatesare shown to be effective against tumors, as well as against otherpathophysiological conditions caused by a proliferation of cells whichare sensitive to FGF mitogenic stimulation.

[0306] 2. Treatment of SMC Disorders

[0307] The conjugates may be used to treat or prevent atherosclerosisand stenosis, a process and the resulting condition that occursfollowing angioplasty in which the arteries become reclogged. Generally,treatment of atherosclerosis involves widening a stenotic vascularlumen, permitting greater blood flow and oxygenation to the distaltissue. Unfortunately, these procedures induce a normal wound healingresponse in the vasculature that results in restenosis. Of the threecomponents to the normal vascular response to injury, thrombosis,elastic recoil and smooth muscle cell proliferation,anti-thrombotics/platelet inhibitors and vascular stents effectivelyaddress acute/subacute thrombosis and elastic recoil, respectively.However, no therapy can modify the vascular remodeling that is due toproliferation of smooth muscle cells at the lesion, their deposition ofextracellular matrix and the subsequent formation of a neointima.Accordingly, restenosis remains a significant clinical problem.

[0308] Wound response also occurs after other interventions, such asballoon angioplasty of coronary and peripheral vessels, with or withoutstenting; carotid endarterectomies; vein grafts; and synthetic grafts inperipheral arteries and arteriovenous shunts. Although the time courseof the wound response is not well defined, if the response can besuppressed for a short term (approximately 2 weeks), a long term benefitis achieved.

[0309] 3. Treatment of Angiogenic Diseases

[0310] As noted above, the compositions of the present invention areused to treat angiogenesis-dependent diseases. In these diseases,vascular growth is excessive or allows unwanted growth of other tissuesby providing blood supply. These diseases include angiofibroma,arteriovenous malformations, arthritis, atherosclerotic plaques, cornealgraft neovascularization, delayed wound healing, diabetic retinopathy,granulations due to burns, hemangiomas, hemophilic joints, hypertrophicscars, neovascular glaucoma, nonunion fractures, Osler-weber syndrome,psoriasis, pyogenic granuloma, retrolental fibroplasia, scleroderma,solid tumors, trachoma, and vascular adhesions.

[0311] By inhibiting vessel formation (angiogenesis), unwanted growthmay be slowed or halted, thus ameliorating the disease. In a normalvessel, a single layer of endothelial cells lines the lumen. Growth of avessel requires proliferation of endothelial cells and smooth musclecells. As such, the present invention provides nucleic acid deliveryvehicles that bind to cell surface molecules (receptors) via a ligandand internalize, thus delivering a nucleic acid molecule.

[0312] 4. Positive Gene Therapy

[0313] The molecules, constructs and methods of the present inventionmay also be useful in a wide variety of so-called “positive genetherapy” applications. Since positive gene therapy applications havebeen discussed in detail in earlier sections of the specification, thatinformation will not be repeated herein. Nevertheless, it should beapparent to one of skill in the art that the molecules, constructs andmethods of the present invention are able to effect a treatment upon orwithin a cell, generally by modifying gene transcription of translation,which makes them ideal in a variety of “positive” gene therapyapplications, such as the stimulation of wound repair and bone regrowth.

[0314] A wide variety of positive gene therapy applications andtherapeutic gene products have thus been described above and includesuch diverse applications as the treatment of ischemia, the promotion ofwound healing, the stimulation of bone growth and regrowth, increasedvascularization, and the like. The augmentation or replacement of a“defective” or nonfunctional gene with one that produces the desiredgene product is also considered “positive” gene therapy, whether one isreplacing a dysfunctional or nonfunctional regulatory sequence or asequence that encodes a structural protein.

[0315] 5. Preparation of Pharmaceutical Agents

[0316] Pharmaceutical carriers or vehicles suitable for administrationof the conjugates and complexes provided herein include any suchcarriers known to those skilled in the art to be suitable for theparticular mode of administration. In addition, the conjugates andcomplexes may be formulated as the sole pharmaceutically activeingredient in the composition or may be combined with other activeingredients.

[0317] The conjugates and complexes can be administered by anyappropriate route, for example, orally, parenterally, includingintravenously, intradermally, subcutaneously, or topically, in liquid,semi-liquid or solid form and are formulated in a manner suitable foreach route of administration. Preferred modes of administration dependupon the indication treated. Dermatological and ophthalmologicindications will typically be treated locally; whereas, tumors andrestenosis, will typically be treated by systemic, intradermal, orintramuscular modes of administration.

[0318] The conjugates and complexes herein may be formulated intopharmaceutical compositions suitable for topical, local, intravenous andsystemic application. For ophthalmic uses, local administration, eitherby topical administration or by injection is preferred.

[0319] Time release formulations are also desirable, irrespective of theroute or form in which the conjugates and complexes of the presentinvention are administered. Effective concentrations of one or more ofthe conjugates and complexes are mixed with a suitable pharmaceuticalcarrier or vehicle. As used herein an “effective amount” of a compoundfor treating a particular disease is an amount that is sufficient toameliorate, or in some manner reduce the symptoms associated with thedisease. Such amount may be administered as a single dosage or may beadministered according to a regimen, whereby it is effective. The amountmay cure the disease but, typically, is administered in order toameliorate the symptoms of the disease. Repeated administration may berequired to achieve the desired amelioration of symptoms.

[0320] As used herein, “an effective amount” is that amount which, inthe composition administered and by the technique administered, providesan amount of therapeutic agent to the involved tissues sufficient toprevent or reduce cell proliferation or to ameliorate quiescent ormetabolic disease.

[0321] The concentrations or amounts of the conjugates and complexesthat are effective requires delivery of an amount, upon administration,that ameliorates the symptoms or treats the disease. Typically, thecompositions are formulated for single dosage administration.Therapeutically effective concentrations and amounts may be determinedempirically by testing the conjugates and complexes in known in vitroand in vivo systems, such as those described here; dosages for humans orother animals may then be extrapolated therefrom.

[0322] The conjugate is included in the pharmaceutically acceptablecarrier in an amount sufficient to exert a therapeutically useful effectin the absence of undesirable side effects on the patient treated. Theconjugates may be delivered as pharmaceutically acceptable salts, estersor other derivatives of the conjugates include any salts, esters orderivatives that may be readily prepared by those of skill in this artusing known methods for such derivatization and that produce compoundsthat may be administered to animals or humans without substantial toxiceffects. It is understood that number and degree of side effects dependsupon the condition for which the conjugates and complexes areadministered. For example, certain toxic and undesirable side effectsare tolerated when treating life-threatening illnesses, such as tumors,that would not be tolerated when treating disorders of lesserconsequence. The concentration of conjugate in the composition willdepend on absorption, inactivation and excretion rates thereof, thedosage schedule, and amount administered as well as other factors knownto those of skill in the art.

[0323] Preferably, the conjugate and complex are substantially pure. Asused herein, “substantially pure” means sufficiently homogeneous toappear free of readily detectable impurities as determined by standardmethods of analysis, such as thin layer chromatography (TLC), gelelectrophoresis, high performance liquid chromatography (HPLC), used bythose of skill in the art to assess such purity, or sufficiently puresuch that further purification would not detectably alter the physicaland chemical properties, such as enzymatic and biological activities, ofthe substance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound may, however, be amixture of stereoisomers. In such instances, further purification mightincrease the specific activity of the compound.

[0324] The conjugates and complexes may be formulated for local ortopical application, such as for topical application to the skin andmucous membranes, such as in the eye, in the form of gels, creams, andlotions and for application to the eye or for intracistemal orintraspinal application. Such solutions, particularly those intended forophthalmic use, may be formulated as 0.01%-10% isotonic solutions, pHabout 5-7, with appropriate salts. The ophthalmic compositions may alsoinclude additional components, such as hyaluronic acid. The conjugatesand complexes may be formulated as aerosols for topical application(see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923).

[0325] Solutions or suspensions used for parenteral, intradermal,subcutaneous, or topical application can include any of the followingcomponents: a sterile diluent, such as water for injection, salinesolution, fixed oil, polyethylene glycol, glycerine, propylene glycol orother synthetic solvent; antimicrobial agents, such as benzyl alcoholand methyl parabens; antioxidants, such as ascorbic acid and sodiumbisulfite; chelating agents, such as ethylenediaminetetraacetic acid(EDTA); buffers, such as acetates, citrates and phosphates; and agentsfor the adjustment of toxicity such as sodium chloride or dextrose.Parental preparations can be enclosed in ampules, disposable syringes ormultiple dose vials made of glass, plastic or other suitable material.

[0326] If administered intravenously, suitable carriers includephysiological saline or phosphate buffered saline (PBS), and solutionscontaining thickening and solubilizing agents, such as glucose,polyethylene glycol, and polypropylene glycol and mixtures thereof.Liposomal suspensions may also be suitable as pharmaceuticallyacceptable carriers. These may be prepared according to methods known tothose skilled in the art.

[0327] Upon mixing or addition of the conjugate(s) with the vehicle, theresulting mixture may be a solution, suspension, emulsion or the like.The form of the resulting mixture depends upon a number of factors,including the intended mode of administration and the solubility of theconjugate in the selected carrier or vehicle. The effectiveconcentration is sufficient for ameliorating the symptoms of thedisease, disorder or condition treated and may be empirically determinedbased upon in vitro and/or in vivo data, such as the data from the mousexenograft model for tumors or rabbit ophthalmic model. If necessary,pharmaceutically acceptable salts or other derivatives of the conjugatesand complexes may be prepared.

[0328] The active materials can also be mixed with other activematerials, that do not impair the desired action, or with materials thatsupplement the desired action, including viscoelastic materials, such ashyaluronic acid, which is sold under the trademark HEALON (solution of ahigh molecular weight (MW of about 3 millions) fraction of sodiumhyaluronate; manufactured by Pharmacia, Inc. see, e.g., U.S. Pat. Nos.5,292,362, 5,282,851, 5,273,056, 5,229,127, 4,517,295 and 4,328,803),VISCOAT fluorine-containing (meth)acrylates; suchas,1H,1H,2H,2H-hepta-decafluorodecylmethacrylate; see, e.g., U.S. Pat.Nos. 5,278,126, 5,273,751 and 5,214,080; commercially available fromAlcon Surgical, Inc.), ORCOLON (see, e.g., U.S. Pat. Nos. 5,273,056;commercially available from Optical Radiation Corporation),methylcellulose, methyl hyaluronate, polyacrylamide andpolymethacrylamide (see, e.g., U.S. Pat. No. 5,273,751). Theviscoelastic materials are present generally in amounts ranging fromabout 0.5 to 5.0%, preferably 1 to 3% by weight of the conjugatematerial and serve to coat and protect the treated tissues. Thecompositions may also include a dye, such as methylene blue or otherinert dye, so that the composition can be seen when injected into theeye or contacted with the surgical site during surgery.

[0329] The conjugates and complexes may be formulated for local ortopical application, such as for topical application to the skin andmucous membranes, such as in the eye, in the form of gels, creams, andlotions and for application to the eye. Such solutions, particularlythose intended for ophthalmic use, may be formulated as 0.01%-10%isotonic solutions, pH about 5-7, with appropriate salts. Suitableophthalmic solutions are known (see, e.g., U.S. Pat. No. 5,116,868,which describes typical compositions of ophthalmic irrigation solutionsand solutions for topical application). Such solutions, which have a pHadjusted to about 7.4, contain, for example, 90-100 mM sodium chloride,4-6 mM dibasic potassium phosphate, 4-6 mM dibasic sodium phosphate,8-12 mM sodium citrate, 0.5-1.5 mM magnesium chloride, 1.5-2.5 mMcalcium chloride, 15-25 mM sodium acetate, 10-20 mM D.L.-sodiumβ-hydroxybutyrate and 5-5.5 mM glucose.

[0330] The conjugates and complexes may be prepared with carriers thatprotect them against rapid elimination from the body, such as timerelease formulations or coatings. Such carriers include controlledrelease formulations, such as, but not limited to, implants andmicroencapsulated delivery systems, and biodegradable, biocompatiblepolymers, such as carboxymethylcellulose, ethylene vinyl acetate,polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid andothers. For example, the composition may be applied during surgery usinga sponge, such as a commercially available surgical sponges (see, e.g.,U.S. Pat. Nos. 3,956,044 and 4,045,238; available from Weck, Alcon, andMentor), that has been soaked in the composition and that releases thecomposition upon contact with the eye. These are particularly useful forapplication to the eye for ophthalmic indications following or duringsurgery in which only a single administration is possible. Thecompositions may also be applied in pellets (such as Elvaxpellets—ethylene-vinyl acetate copolymer resin); about 1-5 μg ofconjugate per 1 mg resin) that can be implanted in the eye duringsurgery.

[0331] If oral administration is desired, the conjugate should beprovided in a composition that protects it from the acidic environmentof the stomach. For example, the composition can be formulated in anenteric coating that maintains its integrity in the stomach and releasesthe active compound in the intestine. The composition may also beformulated in combination with an antacid or other such ingredient.

[0332] Oral compositions will generally include an inert diluent or anedible carrier and may be compressed into tablets or enclosed in gelatincapsules. For the purpose of oral therapeutic administration, the activecompound or compounds can be incorporated with excipients and used inthe form of tablets, capsules or troches. Pharmaceutically compatiblebinding agents and adjuvant materials can be included as part of thecomposition.

[0333] The tablets, pills, capsules, troches and the like can containany of the following ingredients, or compounds of a similar nature: abinder, such as microcrystalline cellulose, gum tragacanth and gelatin;an excipient such as starch and lactose, a disintegrating agent such as,but not limited to, alginic acid and corn starch; a lubricant such as,but not limited to, magnesium stearate; a glidant, such as, but notlimited to, colloidal silicon dioxide; a sweetening agent such assucrose or saccharin; and a flavoring agent such as peppermint, methylsalicylate, and fruit flavoring.

[0334] When the dosage unit form is a capsule, it can contain, inaddition to material of the above type, a liquid carrier such as a fattyoil. In addition, dosage unit forms can contain various other materialswhich modify the physical form of the dosage unit, for example, coatingsof sugar and other enteric agents. The conjugates and complexes can alsobe administered as a component of an elixir, suspension, syrup, wafer,chewing gum or the like. A syrup may contain, in addition to the activecompounds, sucrose as a sweetening agent and certain preservatives, dyesand colorings and flavors.

[0335] The active materials can also be mixed with other activematerials that do not impair the desired action, or with materials thatsupplement the desired action, such as cis-platin for treatment oftumors.

[0336] Finally, the compounds may be packaged as articles of manufacturecontaining packaging material, one or more conjugates and complexes orcompositions as provided herein within the packaging material, and alabel that indicates the indication for which the conjugate is provided.

[0337] 6. Administration

[0338] Typically a therapeutically effective dosage should produce aserum concentration of active ingredient of from about 0.1 ng/ml toabout 500 μg/ml. The pharmaceutical compositions typically shouldprovide a dosage of from about 0.01 mg/kg to about 100-2000 mg/kg ofconjugate, depending upon the conjugate. Local application forophthalmic disorders and dermatological disorders should provide about 1ng up to 100 μg, preferably about 1 ng to about 10 μg, per single dosageadministration. It is understood that the amount to administer will be afunction of the conjugate selected, the indication treated, and possiblythe side effects that will be tolerated.

[0339] Therapeutically effective concentrations and amounts may bedetermined for each application herein empirically by testing theconjugates and complexes in known in vitro and in vivo systems (e.g.,murine, rat, rabbit, or baboon models), such as those described herein;dosages for humans or other animals may then be extrapolated therefrom.The rabbit eye model is a recognized model for studying the effects oftopically and locally applied drugs (see, e.g., U.S. Pat. Nos.5,288,735, 5,263,992, 5,262,178, 5,256,408, 5,252,319, 5,238,925,5,165,952; see also Mirate et al., Curr. Eye Res. I:491-493, 1981).

[0340] The active ingredient may be administered at once, or may bedivided into a number of smaller doses to be administered at intervalsof time. It is understood that the precise dosage and duration oftreatment is a function of the disease being treated and may bedetermined empirically using known testing protocols or by extrapolationfrom in vivo or in vitro test data. It is to be noted thatconcentrations and dosage values may also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed compositions.

[0341] 7. Therapeutic Sequences and Compositions

[0342] A therapeutic nucleotide composition of the present inventioncomprises a nucleotide sequence encoding a therapeutic molecule asdescribed herein. As noted above, a therapeutic nucleotide compositionmay further comprise an enhancer element or a promoter located 5′ to andcontrolling the expression of said therapeutic nucleotide sequence orgene. The promoter is a DNA segment that contains a DNA sequence thatcontrols the expression of a gene located 3′ or downstream of thepromoter. The promoter is the DNA sequence to which RNA polymerasespecifically binds and initiates RNA synthesis (transcription) of thatgene, typically located 3′ of the promoter.

[0343] The subject therapeutic nucleotide composition consists of anucleic acid molecule that comprises at least 2 different operativelylinked DNA segments. The DNA can be manipulated and amplified by PCR andby using the standard techniques described in Molecular Cloning: ALaboratory Manual, 2nd Edition, Maniatis et al., eds., Cold SpringHarbor, N.Y. (1989). Typically, to produce a therapeutic nucleotidecomposition of the present invention, the sequence encoding the selectedtherapeutic composition and the promoter or enhancer are operativelylinked to a vector DNA molecule capable of autonomous replication in acell either in vivo or in vitro. By operatively linking the enhancerelement or promoter and the nucleotide sequence encoding the therapeuticnucleotide composition to the vector, the attached segments arereplicated along with the vector sequences. Thus, a recombinant DNAmolecule (rDNA) of the present invention is a hybrid DNA moleculecomprising at least 2 nucleotide sequences not normally found togetherin nature.

[0344] The therapeutic nucleotide composition of the present inventionis from about 20 base pairs to about 100,000 base pairs in length.Preferably the nucleic acid molecule is from about 50 base pairs toabout 50,000 base pairs in length. More preferably the nucleic acidmolecule is from about 50 base pairs to about 10,000 base pairs inlength. Most preferred is a nucleic acid molecule from about 50 pairs toabout 4,000 base pairs in length. The therapeutic nucleotide can be agene or gene fragment that encodes a protein or peptide that providesthe desired therapeutic effect such as replacement of alpha1-antitrypsin or cystic fibrosis transmembrane regulator protein and thelike. Alternatively, the therapeutic nucleotide can be a DNA or RNAoligonucleotide sequence that exhibits enzymatic therapeutic activity.Examples of the latter include antisense oligonucleotides that willinhibit the transcription of deleterious genes or ribozymes that act assite-specific ribonucleases for cleaving selected mutated genesequences. In another variation, a therapeutic nucleotide sequence ofthe present invention may comprise a DNA construct capable of generatingtherapeutic nucleotide molecules, including ribozymes and antisense DNA,in high copy numbers in target cells, as described in published PCTapplication No. WO 92/06693 (the disclosure of which is incorporatedherein by reference).

[0345] A regulatable promoter is a promoter where the rate of RNApolymerase binding and initiation is modulated by external stimuli. Suchstimuli include compositions light, heat, stress and the like.Inducible, suppressible and repressible promoters are regulatablepromoters. Regulatable promoters may also include tissue specificpromoters. Tissue specific promoters direct the expression of that geneto a specific cell type. Tissue specific promoters cause the genelocated 3′ of it to be expressed predominantly, if not exclusively inthe specific cells where the promoter expressed its endogenous gene.Typically, it appears that if a tissue-specific promoter expresses thegene located 3′ of it at all, then it is expressed appropriately in thecorrect cell types as has been reviewed by Palmiter et al., Ann. Rev.Genet. 20: 465-499 (1986).

[0346] When a tissue specific promoter controls the expression of agene, that gene will be expressed in a small number of tissues or celltypes rather than in substantially all tissues and cell types. Examplesof tissue specific promoters include the immunoglobulin promoterdescribed by Brinster et al., Nature 306: 332-336 (1983) and Storb etal., Nature 310: 238-231 (1984); the elastase-I promoter described bySwift et al., Cell 38: 639-646 (1984); the globin promoter described byTownes et al., Mol. Cell. Biol. 5: 1977-1983 (1985), and Magram et al.,Mol. Cell. Biol. 9: 4581-4584 (1989), the insulin promoter described byBucchini et al., PNAS USA 83: 2511-2515 (1986) and Edwards et al., Cell58: 161 (1989); the immunoglobulin promoter described by Ruscon et al.,Nature 314: 330-334 (1985) and Grosscheld et al., Cell 38: 647-658(1984); the alpha actin promoter described by Shani, Mol. Cell. Biol. 6:2624-2631 (1986); the alpha crystalline promoter described by Overbeeket al., PNAS USA 82: 7815-7819 (1985); the prolactin promoter describedby Crenshaw et al., Genes and Development 3: 959-972 (1989); theproopiomelanocortin promoter described by Tremblay et al.,_(—) PNAS USA85: 8890-8894 (1988); the beta-thyroid stimulating hormone (BTSH)promoter described by Tatsumi et al., Nippon Rinsho 47: 2213-2220(1989); the mouse mammary tumor virus (MMTV) promoter described byMuller et al., Cell 54: 105 (1988); the albumin promoter described byPalmiter et al., Ann. Rev. Genet. 20: 465-499 (1986); the keratinpromoter described by Vassar et al., PNAS USA 86: 8565-8569 (1989); theosteonectin promoter described by McVey et al., J. Biol. Chem. 263:11,111-11,116 (1988); the prostate-specific promoter described byAllison et al., Mol. Cell. Biol. 9: 2254-2257 (1989); the opsin promoterdescribed by Nathans et al., PNAS USA 81: 4851-4855 (1984); theolfactory marker protein promoter described by Danciger et al., PNAS USA86: 8565-8569 (1989); the neuron-specific enolase (NSE) promoterdescribed by Forss-Pelter et al., J. Neurosci. Res. 16: 141-151 (1986);the L-7 promoter described by Sutcliffe, Trends in Genetics 3: 73-76(1987) and the protamine 1 promoter described Peschon et al., Ann. NewYork Acad. Sci. 564: 186-197 (1989) and Braun et al., Genes andDevelopment 3: 793-802 (1989).

[0347] In various alternative embodiments of the present invention,therapeutic sequences and compositions useful for practicing thetherapeutic methods described herein are contemplated. Therapeuticcompositions of the present invention may contain a physiologicallytolerable carrier together with one or more therapeutic nucleotidesequences of this invention, dissolved or dispersed therein as an activeingredient. In a preferred embodiment, the composition is notimmunogenic or otherwise able to cause undesirable side effects whenadministered to a mammal or human patient for therapeutic purposes.

[0348] As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike.

[0349] Compositions designed to preferentially target non-epithelialcells may include an adenovirus-derived protein-ligand conjugate and atherapeutic nucleotide sequence. Examples of useful ligands directed tospecific receptors (identified in parentheses) include FGF and relatedligands (FGFR); the V3 loop of HIV gp120 (CD4); transferrin (transferrinreceptor); LDL (LDL receptors); and deglycosylated proteins(asialoglycoprotein receptor). Polypeptides having a sequence thatincludes an amino acid residue sequence selected from the groupcomprising -EDPGFFNVE- and -EDPGKQLYNVE- are capable of targetingreceptors such as the CR2 receptor, and are thus useful in compositionsdisclosed herein.

[0350] Useful ligands also include antibodies and attachment sequences,as well as receptors themselves. Antibodies to cell receptor moleculessuch as integrins and the like, MHC Class I and Class II,asialoglycoprotein receptor, transferrin receptors, LDL receptors, CD4,and CR2 are but a few useful according to the present invention. It isalso understood that the ligands typically bound by receptors, as wellas analogs to those ligands, may be used as cellular targeting agents asdisclosed herein.

[0351] Exemplary and preferred nucleotide sequences encode anexpressible peptide, polypeptide or protein, and may further include anactive constitutive or inducible promoter sequence. For example,preferred therapeutic nucleotide sequences according to the presentinvention are capable of delivering HIV antisense nucleotides tolatently-infected T cells via CD4. Similarly, delivery of Epstein-BarrVirus (EBV) EBNa-1 antisense nucleotides to B cells via CR2 is capableof effecting therapeutic results.

[0352] The preparation of a pharmacological composition that containsactive ingredients dissolved or dispersed therein is well understood inthe art. Typically such compositions are prepared as injectables eitheras liquid solutions or suspensions, however, solid forms suitable forsolution, or suspensions, in liquid prior to use can also be prepared.The preparation can also be emulsified, or formulated intosuppositories, ointments, creams, dermal patches, or the like, dependingon the desired route of administration.

[0353] The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. Suitable excipients are, for example, water, saline, dextrose,glycerol, ethanol or the like and combinations thereof, includingvegetable oils, propylene glycol, polyethylene glycol and benzyl alcohol(for injection or liquid preparations); and vaseline, vegetable oil,animal fat and polyethylene glycol (for externally applicablepreparations). In addition, if desired, the composition can containwetting or emulsifying agents, isotonic agents, dissolution promotingagents, stabilizers, colorants, antiseptic agents, soothing agents andthe like additives (as usual auxiliary additives to pharmaceuticalpreparations), pH buffering agents and the like which enhance theeffectiveness of the active ingredient.

[0354] The therapeutic compositions of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

[0355] Physiologically tolerable carriers are well known in the art.Exemplary of liquid carriers are sterile aqueous solutions that containno materials in addition to the active ingredients and water, or containa buffer such as sodium phosphate at physiological pH value,physiological saline or both, such as phosphate-buffered saline. Stillfurther, aqueous carriers can contain more than one buffer salt, as wellas salts such as sodium and potassium chlorides, dextrose, polyethyleneglycol and other solutes.

[0356] Liquid compositions can also contain liquid phases in addition toand to the exclusion of water. Exemplary of such additional liquidphases are glycerin, vegetable oils such as cottonseed oil, andwater-oil emulsions.

[0357] A therapeutic composition typically contains an amount of atherapeutic nucleotide sequence of the present invention sufficient todeliver a therapeutically effective amount to the target tissue,typically an amount of at least 0.1 weight percent to about 90 weightpercent of therapeutic nucleotide sequence per weight of totaltherapeutic composition. A weight percent is a ratio by weight oftherapeutic nucleotide sequence to total composition. Thus, for example,0.1 weight percent is 0.1 grams of DNA segment per 100 grams of totalcomposition.

[0358] The therapeutic nucleotide compositions comprising syntheticoligonucleotide sequences of the present invention can be prepared usingany suitable method, such as, the phosphotriester or phosphodiestermethods. See Narang et al., Meth. Enzymol. 68: 90, (1979); U.S. Pat. No.4,356,270; and Brown et al., Meth. Enzymol. 68 :109, (1979). Fortherapeutic oligonucleotides sequence compositions in which a family ofvariants is preferred, the synthesis of the family members can beconducted simultaneously in a single reaction vessel, or can besynthesized independently and later admixed in preselected molar ratios.

[0359] For simultaneous synthesis, the nucleotide residues that areconserved at preselected positions of the sequence of the family membercan be introduced in a chemical synthesis protocol simultaneously to thevariants by the addition of a single preselected nucleotide precursor tothe solid phase oligonucleotide reaction admixture when that positionnumber of the oligonucleotide is being chemically added to the growingoligonucleotide polymer. The addition of nucleotide residues to thosepositions in the sequence that vary can be introduced simultaneously bythe addition of amounts, preferably equimolar amounts, of multiplepreselected nucleotide precursors to the solid phase oligonucleotidereaction admixture during chemical synthesis. For example, where allfour possible natural nucleotides (A,T,G and C) are to be added at apreselected position, their precursors are added to the oligonucleotidesynthesis reaction at that step to simultaneously form four variants.

[0360] This manner of simultaneous synthesis of a family of relatedoligonucleotides has been previously described for the preparation of“degenerate oligonucleotides” by Ausubel et al, in Current Protocols inMolecular Biology, Suppl. 8, p.2.11.7, John Wiley & Sons, Inc., New York(1991), and can readily be applied to the preparation of the therapeuticoligonucleotide compositions described herein.

[0361] Nucleotide bases other than the common four nucleotides (A,T,G orC), or the RNA equivalent nucleotide uracil (U), can be used in thepresent invention. For example, it is well known that inosine (I) iscapable of hybridizing with A, T and G, but not C. Thus, where all fourcommon nucleotides are to occupy a single position of a family ofoligonucleotides, that is, where the preselected therapeutic nucleotidecomposition is designed to contain oligonucleotides that can hybridizeto four sequences that vary at one position, several differentoligonucleotide structures are contemplated. The composition can containfour members, where a preselected position contains A,T,G or C.Alternatively, the composition can contain two members, where apreselected position contains I or C, and has the capacity the hybridizeat that position to all four possible common nucleotides. Finally, othernucleotides may be included at the preselected position that have thecapacity to hybridize in a non-destabilizing manner with more than oneof the common nucleotides in a manner similar to inosine.

[0362] 8. Testing of Constructs

[0363] The reprogrammed viral delivery vehicles may be assessed in anynumber of in vitro model systems. In particular, target cells are grownin culture and incubated with the nucleic acid delivery vehicle. Thenucleic acid can encode a reporter, in which case, the reporter productis assayed, or encode a cytocidal product, in which case cell killing ismeasured. Moreover, any assayable gene product can be used. For reportergenes, a wide variety of suitable genes are available. Such reportersinclude β-galactosidase, alkaline phosphatse, β-glucuronidase, large Tantigen, any protein for which an antibody exists or can be developed.The choice of a reporter depends, in part, upon the cells being tested.Alternatively, the nucleic acid can encode a cytocidal product. Suchproducts include all those described herein.

[0364] The delivery vehicles may be assessed in in vivo model systems.Generally, a xenogeneic tumor model system will be used, but other tumormodel systems are useful as well. In the xenogeneic system, animmunodeficient mouse, or other immunodeficient animal, is injected withtumor cells, such as human tumor cells. The nucleic acid deliveryvehicle is administered and tumor growth is monitored. Any reduction oftumor growth is useful within the context of this invention.

[0365] The following examples are included for illustrative purposesonly and are not intended to limit the scope of the invention.

EXAMPLES Example 1 Targeted Gene Delivery to Kaposi's Sarcoma Cells

[0366] Infection with human immunodeficiency virus (HIV) is associatedwith an increased incidence of a characteristic subset of neoplasticdisorders including Kaposi's sarcoma (KS) and non-Hodgkin's lymphoma(Conant, Recent Results in Cancer Research 139:423-32 (1995)). In thisregard, KS is the major AIDS-associated malignancy and leAd5 tosignificant morbidity (Conant, Id. (1995); Northfelt and Volberding,Advances in Oncology 7:9-17 (1991)). Effective treatment for KS iscurrently lacking, with the duration of survival being only 9.9 monthswith some newer experimental protocols (Gill, et al., J. Clin. Oncol.14:2353-64 (1996)). thus, the development of novel, more effectivetherapies is required for HIV-associated KS.

[0367] Toward this end, various of gene therapy approaches have beendeveloped for neoplastic diseases (Ross, et al., Hum. Gene Ther.7:1781-90 (1996)). Practical implementation of a gene therapy approachfor KS would require efficient in vivo transduction of the tumor cells,Further, some level of targeting to KS spindle cells would likewise bean important criterion for vector selection. This consideration isespecially relevant in AIDS-related disseminated KS, as this tumor isthought to arise from vascular endothelial cells that are continuouswith the systemic vasculature (Northfelt et al., Id. (1991)). Furthercomplicating this endeavor, if has previously been noted that KS cellsare refractory to transduction by a variety of viral and non-viralvector systems, thus limiting even those gene therapy approaches basedon loco-regional gene delivery. To address this issue, a derivativevector has now been developed which possesses the capacity to target KScells and is further described hereinbelow.

[0368] A. Materials and Methods

[0369] 1. Cell Lines

[0370] The human AIDS-KS cell line KSY-1 (Lunardi-Iskandar, et al., J.Natl. Cancer Inst. 87:974-981 (1995)), RW376, and CVU-1 were obtainedfor use as described herein. KS-SLK (Siegal, et al., Cancer 65:492-498(1990)) was derived from an oral KS lesion in an immunosuppressedpatient and was also obtained for use as described below.

[0371] All cell lines are grown in Dulbecco's Modified Eagle sMedium/Ham s F12 at 1:1 ratio by weight (DMEM/F12 Cellgro Mediatech,Washington, D.C.)+10% fetal bovine serum (FBS, Hyclone, Logan, Utah)+2mM glutamine (Cellgro Mediatech)+penicillin/streptomycin (CellgroMediatech) at 37° C. in 5% CO₂ (CM). Media changes are performed every3-4 days. Cells are passaged using Trypsin/EDTA (Cellgro Mediatech) whencells achieved confluency. Viability is determined in confluent cellsexposed to trypsin/EDTA, centrifuged at 800×g in the presence of CM, andcounted using a hemocytometer after trypan blue exclusion. Ganciclovir(GCV; Cytovene) is purchased from Hoffman Laboratories (Nutley, N.J.).Tissue culture plates and flasks were manufactured by Nunclon (Denmark).

[0372] 2. Anti-Knob Antibodies and Fragments

[0373] The procedures for generating and purifying exemplary antibodiesand fragments as disclosed herein are described in a variety ofreferences known to those of skill in the art (e.g., Douglas, et al.,Nature Biotech. 14:1574-1578 (1996)). In general, the procedures may bedescribed as follows.

[0374] To develop a neutralizing anti-knob mAb, hybridomas are generatedby standard techniques after immunization of mice with intact Ad5followed by two rounds of immunization with purified Ad5 knob (native orrecombinant). On the basis of its high affinity binding to recombinantAd5 knob and its ability to neutralize Ad5 infection of HeLa cells (datanot shown), one clone, designated 1D6.14, was chosen for further studyand the mAb is purified from ascites fluid by affinity chromatographyusing an immobilized protein A column.

[0375] Anti-knob mAbs are generated by established methods (see, e.g.,Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring HarborLaboratory, NY (1988)) after immunization of BALB/c mice with Ad5,followed by two rounds of immunization with purified recombinant Ad5knob (see Henry, et al., J. Virol. 68:5239-46 (1994)). Sensitizedlymphocytes are fused with P3-X63-Ag8.653 cells. The reactivity of thehybridoma supernatants with trimeric Ad5 knob is determined in an ELISA.The ability of the hybridoma supernatants to neutralize Ad5 infection isassayed by endpoint CPE.

[0376] The 1D6.14 hybridoma cells are injected into BALB/c mice andascites fluid collected (Harlow and Lane, Id. (1988)). Purification ofthe mAb is performed by affinity chromatography on immobilized protein Ausing an ImmunoPure IgG purification kit (Pierce, Rockford, Ill.). Fabfragments are prepared and purified by digestion of 1D6.14 onimmobilized papain followed by affinity chromatography on immobilizedprotein A, using an ImmunoPure Fab purification kit (Pierce). Afterextensive dialysis against phosphate-buffered saline (PBS), theconcentrations of the purified mAb and Fab fragment are determined usingthe Bio-Rad protein assay (Bio-Rad, Hercules, Calif.).

[0377] For the purposes of developing a targeted adenoviral vector byimmunological methods, it would be preferable to use the Fab fragment ofthe antibody, rather than the intact immunoglobulin. By using the Fabfragment, the two antigen-binding arms of the parent antibody might beprevented from crosslinking different viruses to form large complexesthat might prove refractory to cellular uptake. Intact 1D6.14 isdigested with papain and the Fab fragments are purified. Both the parentantibody, 1D6.14, and the Fab fragment are capable of neutralizingadenovirus infection in a dose-dependent manner, whereas a controlantibody failed to block infection. (See Douglas et al., Id. (1996).)

[0378] 3. Recombinant Adenovirus

[0379] Recombinant E1A-deleted adenovirus (Herz and Gerard, PNAS USA90:2812-1216 (1993)) expressing firefly luciferase (AdCMV-Luc) isutilized as described hereinbelow. An E1-deleted Ad5 vector expressingthe CMV-driven herpes simplex thymidine kinase gene (AdCMVHSVtk) isconstructed using homologous recombination techniques, as previouslyreported (Rosenfeld et al., Clin. Cancer Res. 1:1571-1589 (1995)). AnE1-deleted recombinant adenovirus expressing an enhanced variant ofgreen fluorescent protein (AdCAG-GFPS65T) is also used and has beendescribed previously (Moriyoshi et al., Neuron 16:255-260 (1996)).

[0380] Recombinant adenoviruses are propagated on the permissive 293cell line, purified using a cesium chloride gradient, and subsequentlyplaque titered on 293 cells employing standard methods (Graham andPrevec, in Methods in Mol. Biol. 7: Gene Transfer and ExpressionTechniques, Murray and Walker (eds.), Humana Press, Clifton, 1991, pp.109-129). Virus stocks are stored frozen at −80° C. until use. 4.Fab-FGF2 Molecular Coniugate The Fab-FGF2 conjugate is constructed bylinking modified recombinant basic fibroblast growth factor (FGF2-3;Sosnowski, et al., J. Biol. Chem. 271:33647-33653 (1996)) with the Fabfragment from a blocking monoclonal antibody, 1D6.14, which wasgenerated against adenovirus type 5 (Ad5) knob region (Douglas, et al.,Nature Biotech. 14:1574-1578 (1996)). For conjugation, the Fab isderivatized with the heterobifunctional crosslinking reagent S-2-pyridyldisulfide (SPDP; Pharmacia, Uppsala, Sweden) at a 1:3 molar ratio andincubated at room temperature for 30 minutes to yield a modified Fabfragment (PDP-Fab).

[0381] The PDP-Fab is dialyzed to remove unbound linker. Purified FGF2is generated as previously described (Sosnowski et al., Id. (1996)),then reduced and mixed at a 2:1 molar ratio with PDP-Fab, and incubatedat 4° C. for 16 hrs with shaking.

[0382] In general, FGF2 is prepared and reduced as follows. A155-amino-acid human FGF2, in which the cysteine at position 96 ismutagenized to serine (Lappi, D. A., Matsunami, R., Martineau, D., andBaird, A. (1993) Anal. Biochem. 212:446-451), may be used as describedin the present invention. It should be appreciated that this molecule isdescribed as exemplary, and not as a limitation; other variants of FGFand polypeptides reactive with the FGF receptor complex are usefulaccording to the present invention.

[0383] FGF2 is expressed in E. coli, and purified to homogeneity byconventional chromatography techniques. FGF2 (C96S; may also be referredto herein as FGF2-3) is adjusted to pH 7.0 by adding Tris-base. FGF2 isthen reduced by adding MTG to a final concentration of 20 mM. Thereaction is allowed to incubate at room temperature for 30 minutes.Excess MTG is removed by passing FGF2 (C96S) over a PD-10 column(Pharmacia). Running buffer is 10 mM NaOAc/HOAc pH 5.4 containing 0.14 MNaCl, 1 mM EDTA.

[0384] The Fab is thiolated essentially as follows. 1.6 mg of Fab isdialyzed against NaPO4 (0.1 M Sodium Phosphate buffer, pH 7.5 containing0.1 M NaCl and 1.0 mM EDTA) at 1:250 (v/v) for 3 hr with 2 changes ofbuffer. The dialyzed Fab fragment is centrifuged at 14,000 rpm(Eppendorf centrifuge 5415C) for 10 minutes and the supernatantcollected. The Fab fragment is derivitized with SPDP (Pharmacia), (SPDPdissolved in ethanol), at a molar ratio of 1:3 for 30 minutes at roomtemperature with occasional stirring. The excess SPDP and low molecularweight reaction products are removed by dialysis against the bufferdescribed above at 1:500 (v/v).

[0385] Conjugation of FGF2 and the Fab is carried out essentially asfollows. FGF2 and PDP-Fab are mixed at a molar ratio of 2:1 at pH 7.5and incubated at 4 C for 16 hours with shaking. An aliquot of thereaction mixture is analysed by SEC-HPLC. The conjugate is purified overa Heparin-Sepharose column (1 ml Heparin Hi-Trap, Pharmacia) to removeunconjugated Fab fragment. The material is loaded onto the column in 10mM Tris pH 7.4 and washed in the same buffer plus 0.6 M NaCl. When theabsorbance returns to background the conjugate is eluted from the columnin the same buffer containing 2 M NaCl. An aliquot of the 2M eluate isanalyzed by SEC-HPLC. The 2M eluate is loaded onto Sephacryl S-100 toremove free FGF2 and buffer exchanged into PBS, pH 7.4. Fractions 17-26are pooled as final purified Fab-FGF2 material.

[0386] The conjugation reaction is monitored by reducing an aliquot ofthe reaction mixture with DTT and monitoring the absorbance of PDP at343 nM. Purified Fab-FGF2, FGF2 and Fab are analyzed by SDS-PAGE (12%)and by Western analysis using an antibody generated against FGF2. Todetermine if conjugation to the Fab interfered with FGF2's ability tobind to the receptor and stimulate proliferation, the material isassayed in an endothelial proliferation assay. Bovine aortic endothelialcells are seeded at 1000 cells/well on a 24 well flat-bottom tissueculture plate in DMEM (Biowhittaker), 10% FCS (Hyclone), 50 mg/mlGentamycin (JRH Biosciences), and 2 mM L-glutamine (Biowhittaker). Thefollowing day serial dilutions of FGF2 and Fab-FGF2 ranging from 6 ng/mlto 10 pg/ml, are added to the wells in triplicate. After 48 hours themedia is removed and 1.5 mls of fresh media containing the sameconcentrations of FGF2 and Fab-FGF2 are added to the cells. Followinganother 72 hours of incubation the media is removed, the cells arewashed with PBS and then harvested with 0.25% trypsin. The trypsinizedcells are counted using a Coulter Counter. The results of theproliferation assay reveal that conjugation of the Fab fragment to FGF2did not interfere with FGF2's ability to bind to its receptor andstimulate proliferation.

[0387] The conjugate is purified over a heparin-Sepharose column(Pharmacia) by loading in 10 mM Tris HCl, pH 7.4, washing with 10 mMTris HCl/0.6 mM NaCl, pH 7.4 and eluting in 10 mM Tris HCl/2M NaCl, pH7.4. The eluant is separated over a Sephacryl S-100 column equilibratedwith Dulbecco s phosphate-buffered saline (PBS, pH 7.4) to remove excesssalt and unconjugated protein. The presence of PDP in the conjugate isconfirmed by reducing an aliquot of the conjugate and measuring theabsorbance of PDP (342 nanometers). The size and activity of theconjugate is subsequently analyzed by western blot (Immunoblotting, inAntibodies: A Laboratory Manual, Chapter 12, Harlow and Lane (eds.),Cold Spring Harbor Laboratory (1988)) and enzyme-linked immunoassay(ELISA) analysis (see Immunoassays, in Antibodies: A Laboratory Manual,Chapter 14, Harlow and Lane (eds.), Cold Spring Harbor Laboratory(1988)).

[0388] 5. Adenovirus Infection Assays

[0389] To assess adenoviral transduction, 24,000 cells of each KS cellline are plated in triplicate into each well of a 12-well plate in thepresence of 1 ml of CM. The cells are incubated overnight to allow cellsto adhere. Infection complexes are mixed in a final volume of 50ulcontaining: (1) adenovirus (AdCMV-Luc or Ad-CAG-GFPS65T) at 50 plaqueforming units (pfu)/cell; (2) adenovirus+Fab-FGF2 conjugate; (3)adenovirus+Fab; or (4) adenovirus+Fab-FGF2 conjugate +anti-FGF2 antisera(Sigma), 16ul. The complexes are incubated in 1.5 ml of polypropylenetubes at 27° C. for 30 minutes. The mixtures are then diluted inDMEM/F12+2% FBS and added to each well in a volume of 200 ul. The cellsare incubated at 37° C. in 5% CO₂ for 1 hr, then 800 ul of DMEM/F12+10%FBS is added to each well. Twenty-four hours after the addition ofvirus, the cells are rinsed with PBS and assayed for luciferase activityor analyzed by fluorescence activated cell sorting (FACS). For allluciferase assays, the cells are lysed in 200 ul of Promega (Madison,Wis.) lysis buffer. Twenty ul of each sample is subsequently mixed with100 ul of Promega luciferase assay reagent according to manufacturer sinstructions and triplicate determinations of duplicate samples areassayed in a Berthold luminometer.

[0390] To assess AdCMVHSVtk-mediated killing, 1×10⁵ KSY-1 or KS-SLKcells are plated in duplicate in 6-well plates in 2 ml of CM. The cellsare incubated at 37° C. in 5% CO₂ overnight. The medium is aspirated andinfection mixtures containing 5 pfu/cell of either: (1) AdCMVHSVtk, (2)AdCMVHSVtk +Fab, or (3) AdCMVHSVtk+Fab-FGF2 conjugate are added to eachwell in a volume of 500ul of DMEM/F12+2% FBS. After 1 hr incubation at37° C. in 5% CO₂, 1.5 ml of CM is added. The cells are incubated for anadditional 24 hours and the medium is then aspirated and replaced withCM in the absence (-GCV) or presence (+GCV) of 20 uM GCV. The medium ischanged after 3 days and cell counting is performed in triplicate foreach of the duplicate wells 6 days after exposure to adenovirus toassess TK/GCV-mediated killing.

[0391] 6. Immunocytochemistry

[0392] KS cells (2×10⁴/well) are plated into replicate wells of a24-well tissue culture plate in CM and incubated at 37° C. on 5% CO₂ for48 hrs. The cells are rinsed and endogenous peroxidase is blocked with1% H₂O₂/methanol for 30 minutes. The cells are then rinsed and blockedin 3% bovine serum albumin (BSA; Fraction V, Boehringer Mannheim,Germany)/PBS for 1 hour at 27° C. Rabbit anti-fibroblast growth factorreceptor antiserum (FGFR1- and FGFR2-reactive; Upstate Biotechnologies,Inc., Lake Placid, N.Y.) or control rabbit IgG (Vector; Burlingame,Calif.) is diluted 1:400 in 3% BSA/PBS and allowed to incubate on cellsfor 1 hour at 37° C. The cells are rinsed and stained withdiaminobenzidine (Sigma) using a Vectastain rabbit horseradishperoxidase kit according to the manufacturer's instructions. The cellsare rinsed and stored under water until photomicrographs are taken.

[0393] 7. Statistical Analysis

[0394] A comparison of individual conditions is assessed using Studentst-test for equal means. Statex 1.2 for MacIntosh software (DinanSoftware, Clinton, Iowa) is used to facilitate the analysis.

[0395] B. Results and Discussion

[0396] Gene therapy approaches for KS will depend upon one's ability toaccomplish efficient gene delivery to tumor cells in situ. In thisregard, adenoviral vectors have been employed for a variety of in vivocancer therapy applications. For this application, adenoviral vectorshave the advantage of systemic ability and high levels of geneexpression in vivo.

[0397] Prior to modifying the adenovirus so that it would selectivelyre-target KS cells, the native transduction efficiency of the Ad isexamined. In relevant experiments, two AIDS-KS cell lines (KSY-1 andRW376), one KS cell line from an immunosuppressed patient (KS-SLK), andone classical KS cell line (CVU-1) are employed. In the first set ofexperiments, the adenoviral transduction of each cell line is determinedby infecting each cell line with AdCMV-Luc in the presence or absence ofthe anti-adenovirus knob Fab (see FIG. 1) and subsequently measuringluciferase activity 24 hours after infection.

[0398]FIG. 1 shows a comparison of AdCMV-Luc transduction for four KScell lines. KS cells are incubated with recombinant adenovirusexpressing luciferase in the absence or presence of a Fab fragmentblocking adenoviral knob-mediated infection. Experiments are performedin triplicate. Relative light units (RLU) are shown on the verticalaxis; across the horizontal axis, the following cell lines areindicated: KSY-1; RW376; KS-SLK; and CVU-1. The open (colorless) barrepresents AdCMV-Luc, while the closed (dark) bar represents AdCMV-Luc+anti-knob Fab.

[0399] Of the cell lines tested, KSY-1 and KS-SLK are poorlytransducible by adenovirus, yielding <10⁶ relative light units (RLU) perassay. The KS cell line CVU-1 is moderately transducible(1.83×10⁶+1.15×10⁵ RLU per assay), whereas the RWE376 cell line ishighly transducible, yielding luciferase readings of 2.88×10⁶+5.4×10⁴RLU per assay.

[0400] The luciferase activity obtained after transduction usingAdCMV-Luc correlated with FACS analysis data obtained from cells thatare infected with AdCAG-GFPS65T. In this context, by FACS analysis ofthe KS cell lines transduced with 100 pfu per cell of AdCAG-GFPS65T,fewer than 1% of KSY-1 and KS-SLK cells are transducible. The CVU-1 andTW376 KS cell lines are significantly more transducible yielding 12% and99% transduction efficiencies, respectively. In three cell lines—KSY-1,RW376, and KS-SLK—an anti-adenoviral knob Fab blocked AdCMV-Luctransduction by >50% (p<0.01). The CVU-1 cell line exhibited a lessdramatic (20%)—albeit statistically significant (p<0.05)—block inadenoviral transduction. This low level of inhibition correlates withthe modest level of transduction efficiency by the native adenovirus.This suggests that the degree to which these cells are refractoryinversely correlates with knob-dependent cell binding.

[0401] Based on this recognition, it is hypothesized that thislimitation to infection might be overcome using other cellular entrypathways to achieve effective gene transfer. In this regard, animmunological approach has now been developed that allows retargeting ofadenovirus vectors to heterologous cellular pathways (see Douglas etal., Id. (1996)). As an initial validating step in these studies, wesought to determine whether KS cells expressed FGFR, and whether thisreceptor could serve as a potential substrate for retargeting.

[0402] First, immunocytochemistry is performed on the four KS cell linesusing a polyclonal antibody that simultaneously recognizes FGFR-1 andFGFR-2 via a common epitope. FGFR immunocytochemical reactivity of thefour KS cell lines utilized as described herein is assessed. KSY-1(A,B), RW376 (C,D), KS-SLK (E,F) and CVU-1 (G,H) cell lines are stainedwith polyclonal antiserum raised against a peptide common to FGFR-1 andFGFR-2 or with non-immune control. Immunoreactivity is observed in allfour cell lines (data not shown) as well as in mouse fibroblasts(positive control; not shown). Distribution of immunoreactivity ispredominantly nuclear with scattered cell membrane staining in all fourKS cell lines. The RW376 human KS cell line appeared to have the highestdegree of membrane staining, while the CVU-1 KS line had dense nuclearimmunoreactivity (data not shown). These studies demonstrate that FGFRis highly expressed in the relevant human KS cell lines, consistent withprevious reports (Li, et al., Cancer 72:2253-9 (1993)).

[0403] Once a biologic rationale for the within-described vectorretargeting approach is established, the efficacy of FGFR-targetedadenovirus is then tested using the KS cells as substrates. In a thirdset of experiments, we sought to determine whether we couldimmunologically retarget the adenovirus to FGFR using the Fab as ahandle to the viral knob. To accomplish the retargeting between FGFreceptor and the adenovirus-Fab complex (or conjugate), fibroblastgrowth factor (FGF2) is used as the targeting moiety, since it bindswith high affinity to both FGFR-1 and FGFR-2 and could readily becovalently conjugated to the Fab. Toward this end, a covalent conjugateis synthesized using SDPD to form a disulfide bond between the Fab andthe cysteine present on modified FGF2. Western blot analysis confirmedthat the majority of the Fab-FGF2 conjugate contained a single FGF2molecule and a single Fab fragment. In addition, ELISA-based bindingstudies confirmed that the conjugate simultaneously retainedknob-binding activity and FDGF2 immunoreactivity (data not shown).

[0404] To assess whether the Fab-FGF2 conjugate could retarget theadenovirus to KS cells, the conjugate is first pre-incubated withAdCMV-Luc prior to cellular transduction. In an additional reactionmixture, the AdCMV-Luc+Fab-FGF2 mixture is further incubated withblocking antisera raised against FGFs to assess whether retargeting isoccurring via the FGF2 moiety of the Fab-FGF2 conjugate. FIG. 3illustrates the results of the AdCMV-Luc retargeting experiments usingthe Fab-FGF2 conjugate as well as the FGF2 blocking experiments.

[0405]FIG. 3 shows the enhanced AdCMV-Luc infectivity of KS cell linesby Fab-FGF2 conjugate. The enhanced infectivity of the Ad-conjugatecomplex is assessed in the presence and absence of anti-FGF2 antisera.Relative light units (RLU) are plotted on the vertical axis, while therelevant KS cell lines—KSY-1, RW376, KS-SLK, and CVU-1—are indicated onthe horizontal axis. The closed bars represent AdCMV-Luc; stippled barsrepresent AdCMV-Luc+Fab-FGF2; and the open (colorless) bars representAdCMV-Luc+Fab-FGF2+anti-FGF2 antisera.

[0406] The results shown in FIG. 3 demonstrate a dramatic enhancement ofAdCMV-Luc transduction in all four KS cell lines when the adenovirus ispre-mixed with the Fab-FGF2 conjugate. This unexpected enhancement isstatistically significant for all four cell lines (p<0.001) andrepresents a 44-fold increase in transduction for the KSY-1 cells and a7.7-fold increase for RW376 cells. Of further note, addition of antiseraraised against FGF2 blocked (p<0.01) the ability of the Fab-FGF2conjugate to enhance AdCMV-Luc transduction in all four KS cell lines.The attenuation of conjugate-mediated adenovirus transduction byanti-FGF2 antisera confirmed that retargeting is occurring via the FGFportion of the conjugate.

[0407] The experiments conducted to date demonstrated that the lowtransduction efficiency of the adenovirus accomplished in KS cells couldbe overcome by retargeting the adenovirus to the FGFR pathway. Thedetection of increased luciferase activity confirmed that the transgeneexpression had taken place.

[0408] In an effort to confirm that this paradigm had utility in thecontext of a gene therapy approach whereby a toxin gene is introducedinto KS cells, we performed a series of experiments using a recombinantadenovirus encoding the conditionally toxic gene product, herpes simplexthymidine kinase (AdCMVHSVtk). In this experiment, we chose the two celllines that had demonstrated the highest resistance to adenoviral genetransfer, KSY-1 and KS-SLK. Dose-response killing curves for these twocell lines are generated using cells infected with variousconcentrations of AdMCHSVtk (data not shown) and subsequently maintainedin the presence or absence of GCV. These experiments demonstrated thatboth cell lines showed little evidence of cell killing when cells areinfected with 5 pfu/cell of AdCMVHSVtk in the presence of GCV.

[0409] In subsequent experiments, we sought to potentiate AdCMVHSVtkgene transduction and subsequent sensitization to GCV in KS cells byaddition of the Fab-FGF2 conjugate. In the experimental design, cellsare treated with 5 pfu of either AdCMVHSVtk or AdCMVHSVtk complexed withFab-FGF2. GCV-mediated killing is assessed by maintaining cells in thepresence or absence of GCV. The results of these experiments are shownin FIG. 4.

[0410]FIG. 4 illustrates enhanced AdCMVHSVtk/GCV cell killing in KSY-1and KS-SLK cells by Fab-FGF2 conjugate. The effect of GCV onAdCMVHStk-transfected cells is assessed in the presence or absence ofthe conjugate and expressed as a percentage of cells surviving comparedto the cell not exposed to GCV (i.e., −GCV). Viable cells in duplicatewells are counted, in triplicate, after trypan blue exclusion. On thevertical axis, the % of cells surviving is shown, in both FIGS. 4A and4B. In FIG. 4A, KSY-1 cells transfected with AdCMVHSVtk orAdCMVHSVtk+Fab-FGF2 are identified on the horizontal axis. In FIG. 4B,KS-SLK cells transfected with, AdCMVHSVtk or AdCMVHSVtk+Fab-FGF2 areidentified on the horizontal axis.

[0411] Cell killing is expressed as a ratio of cells surviving in thepresence of GCV relative to the number of cells surviving in the absenceof GCV. FIG. 4 demonstrates that retargeting AdCMVHSVtk with Fab-FGF2resulted in a significant enhancement of the KS cells susceptibility toGCV-mediated killing. These studies thus confirm our hypothesis thatefficient gene transfer may be accomplished in KS cells by retargetingadenovirus via FGFR. Importantly, this maneuver quantitatively increasedtransduction efficiency in all cell lines tested. When Ad does nottarget a cell, this technique allows us to target it to a receptor, andthe resulting response is greater than anticipated.

Example 2 Targeted Gene Delivery via FGFR

[0412] Recombinant adenovirus vectors are of great interest in thecontext of cancer gene therapy due to their ability to accomplishefficient in vivo gene transfer. However, targeting of these vectors tospecific cell types remains an obstacle. To achieve specific targeting,a neutralizing anti-knob antibody fragment (Fab) which inhibits Adinfection is conjugated to the basic fibroblast growth factor (FGF2)ligand. The resulting conjugate, Fab-FGF2, is characterized by Westernanalysis using an anti-FGF2 antibody. Functional validation of the FGF2activity in the conjugate is accomplished using an endothelial cellproliferation assay, and an ELISA is performed to validate that the Fabcomponent of the conjugate still bound to Ad5 knob. The Fab-FGF2conjugate is then used to target an Ad vector carrying the luciferasereporter gene (AdCMVLuc) to FGF receptor-positive cells (Swiss 3T3,PANC-1, SKOV3.1p1, and D54 MG) in vitro.

[0413] Our results demonstrated that the Ad targeted with the Fab-FGF2conjugate achieved a level of gene expression that is significantlygreater than when Ad alone is used in all of the cell lines.Furthermore, the Fab-FGF2 conjugate is able to achieve specific in vivodelivery of AdCMVLuc to SKOV3.1p1 tumors implanted intraperitoneallyinto nude mice. Thus, this work demonstrates that Ad vectors can betargeted to specific cell types in vivo using appropriate ligands. Thisis of tremendous potential utility when using Ad vectors in a variety ofgene therapy strategies.

[0414] C. Materials and Methods

[0415] 1. Cells and Viruses

[0416] PANC-1, a human pancreatic epithelioid carcinoma cell line, andSwiss 3T3, a mouse fibroblast cell line, are obtained from the AmericanType Culture Collection (ATCC, Rockville, Md.). (For example, see ATCCAccession Nos. CRL-1469 and CCL-92, respectively.) D54 MG, human gliomacells, are a derivative of the A172 cell line established by Giard etal. (See, e.g., Giard et al., J. Natl. Cancer Inst. 51:1417-23 (1973);Bigner, et al., J. Neuropathol. Exp. Neurol. 40:390-409 (1981); andGoldman et al., Mol. Biol. Cell 4:121-33 (1993).)

[0417] The SKOV3.1p1 human ovarian adenocarcinoma cell line is kindlyprovided by Janet Price (Baylor University). (The related SKOV3 cellline is available from the ATCC under accession no. HTB-77.) Bovineaortic endothelial cells are obtained from primary cultures(Gospodarowicz, et al., Endocrinology 117:2383-91 (1985)).

[0418] The 3T3, PANC-1 and SKOV3.1p1 cells are maintained in Dulbecco'smodified Eagles medium (DMEM) supplemented with 10% fetal calf serum(FCS) (Summit Biotechnology, Fort Collins, Colo.) and 2 mM L-glutamine.The D54 MG cells are maintained in DMEM/F12 supplemented with 7% FCS and2 mM L-glutamine. The bovine aortic endothelial cells are maintained inDMEM supplemented with 10% FCS, gentamycin (50 ug/mL), 2 mM L-glutamine,1 mM MEM sodium pyruvate solution, and 0.1 mM MEM non-essential aminoacids solution. AdCMVLuc (Herz and Gerard, PNAS USA 90:2812-6 (1993)) isan E1-deleted replication-deficient Ad5 vector which expresses fireflyluciferase (Luc) under the control of the cytomegalovirus (CMV)promoter. The aforementioned vector may be prepared as described in thecited reference. The adenovirus is propagated on the permissive 293 cellline and purified by standard techniques.

[0419] 2. Conjugation of FGF2 to1D6.14-Fab

[0420] The 1D6.14-Fab is generated and characterized as previouslydescribed (Douglas, et al., Nature Biotech. 14:1574-78 (1996)). The Fab(1.6 mg) is dialyzed against 0.1 M sodium phosphate buffer, pH 7.5,containing 0.1 M NaCl and 1.0 mM EDTA (BPS-E) at 1:250 (v/v) for 3 hourswith two changes of buffer. The dialyzed Fab \fragment is centrifuged at14,000 rpm for 10 minutes and the supernatant collected. The Fabfragment is derivatized with N-succinimidyl-3(pyridyldithio) propionate(SPDP) (Pharmacia, Uppsala, Sweden) at a molar ratio of 1:3 for 30minutes at room temperature, with occasional stirring. The excess SPDPand low molecular weight reaction byproducts are removed by dialysisagainst PBS-E (1:500, v/v). An FGF2 mutein is used in all of the studiesdescribed herein; this mutein has its cysteine at position 96mutagenized to serine. The FGF2 mutein is expressed in E. coli andpurified as described previously (Lappi, et al., Anal. Biochem.212:446-51 (1993)).

[0421] The reaction containing the FGF2 mutein is adjusted to pH 7.5 byadding Tris-base and reduced by adding monothioglycerol (MTG; EvansChemetics, Waterloo, N.Y.) to a final concentration of 20 mM. Thereaction is performed at room temperature for 30 minutes before theexcess MTG is removed by passing the mixture over a PD-10 column(Pharmacia) and eluting with 10 mM NaOAc/HOAc, pH 5.4, containing 0.14 MNaCl and 1 mM EDTA. The reduced FGF2 mutein is then mixed with the SPDPderivatized Fab at a molar ratio of 2:1 at pH 7.5 and incubated at 4 Cfor 16 hours with shaking.

[0422] The conjugate is purified over a heparin-Sepharose column (1 mLheparin Hi-Trap, Pharmacia) to remove unconjugated Fab fragment byloading the reaction mixture onto the column in 10 mM Tris pH 7.4 andwashing in the same buffer plus 0.6 M NaCl. When the absorbance returnedto background, the conjugate is eluted from the column with the samebuffer containing 2 M NaCl. The 2 M eluate is then loaded onto aSephacryl S-100 column (Pharmacia) to remove free FGF2 mutein and bufferexchanged into PBS pH 7.4. The final protein concentration of the Fab is0.24 mg/mL. The Fab is used directly in the following studies.

[0423] 3. Characterization of Fab-FGF2 Conjugate

[0424] The Fab-FGF2 conjugate is evaluated by SDS-PAGE (12%) undernonreducing conditions and stained with Coomassie blue. Western blotanalysis is also conducted on the conjugate by transferring protein to anitrocellulose membrane, probing with anti-FGF2 rabbit polyclonalantibodies and then with ¹²⁵I-Protein A, which is revealed byautoradiography.

[0425] The activity of the FGF2 component of the Fab-FGF2 conjugate isconfirmed using a cell proliferation assay. Bovine aortic endothelialcells are seeded at 1000 cells/well in 24-well tissue culture plates.The following day, serial dilutions of FGF2 or the Fab-FGF2 conjugate(30 pg/m: to 6 ng/mL) are added to triplicate wells. After 48 hours, themedia are removed and 1.5 mL of fresh media containing the sameconcentrations of FGF2 and Fab-FGF2 are added. Following another 72 hrincubation, the media are removed, the cells are washed with PBS,treated with 0.25% trypsin, and counted using a Coulter Particle Counter(Coulter).

[0426] The activity of the Fab portion of the Fab-FGF2 conjugate isconfirmed by ELISA. Recombinant trimeric Ad5 knob protein (180 ng) withan N-terminal 6-His tag (Krasnykh, et al., J. Virol. 70:6839-6846(1996)) is plated on Ni-NTA H is Sorb Strips (Qiagen, Chatsworth,Calif.) for 1 hr at room temperature. The Fab-FGF2 conjugate or theappropriate controls are added to the wells and the assay performedaccording to the Qiagen protocol. A polyclonal anti-FGF2 antibody (SigmaImmunochemicals, St. Louis, Mo.) is used as the primary antibody, whilea goat anti-rabbit antibody conjugated to horseradish peroxidase(Southern Biotechnology Associates, Birmingham, Ala.) is used as thesecondary antibody.

[0427] 4. In Vitro Infection Using Fab-FGF2 Conjugate

[0428] 10.5 ug of neutralizing antibody (Fab) or the antibody-conjugate(Fab-FGF2) are incubated with 1.9×108 plaque forming units (pfu) ofAdCMVLuc at room temperature in a total volume of 110 uL of HEPESbuffered saline, pH 7.3. After 30 min, 9 uL of the Fab or Fab-FGF2 ADcomplexes are added in triplicate to the 3T3, PANC-1, SKOV3.1p1, and D54MG cells plated at a density of 24,000 cells/well 24 hours previously.The cells are first washed with PBS and supplemented with 200 uL ofOPTI-MEM reduced serum media (Gibco-BRL, Grand Island, N.Y.) prior toaddition of the complexes. After incubation for 1 hr at 37 C, the cellsare supplemented with 1 mL of complete media and allowed to incubate anadditional 24 hours at 37 C. The cells are then lysed and extractsassayed for luciferase activity using a luciferase assay system(Promega, Madison, Wis.) according to the manufacturer's protocol. Therelative light units (RLU) are normalized for protein content using theBioRad protein assay, following the manufacturer's protocol. Inhibitionstudies are conducted by adding a polyclonal anti-FGF2 antibody (SigmaImmunochemicals) to the AdCMVLuc-Fab-FGF2 mixture prior to cellinfection. All experiments are performed in triplicate.

[0429] 5. In Vivo Infection Using Fab-FGF2 Conjugate

[0430] In vivo experiments are performed in athymic nude mice implantedintraperitoneally (i.p.) with SKOV3.1p1 cells. The SKOV3.1p1 cells(2×107) are implanted via an i.p. injection and allowed to grow for 7days. Mice are then injected i.p. with either AdCMVLuc (1×108 fpu),AdCMVLuc-Fab, AdCMVLuc-Fab-FGF2, or AdCMVLuc-Fab-FGF2 incubated with theanti-FGF2 antibody in a total volume of 600 uL of media containing 2%FCS.

[0431] The AdCMVLuc-Fab and AdCMVLuc-Fab-FGF2 conjugates are made in amanner consistent with that described above. Two days after Adinjection, the mice are sacrificed and the tumors and dorsal mesotheliallining harvested. The organs are rinsed with water, homogenized ingrinding buffer (50 mM K₃PO₄, 1 mM EDTA, 1 mM dithiothreitol, 10%glycerol), and lysed with lysis buffer (Promega). The homogenates areincubated on ice for 30 minutes and then centrifuged for 10 min at14,000 rpm and 4 C. The supernatants are assayed for luciferase activityas described above and standardized for total protein content.

[0432] D. Results and Discussion

[0433] 1. Conjugation of FGF2 to Fab

[0434] The Fab fragment of the ID6.14 anti-Ad5 knob neutralizingantibody is derivatized with SPDP and conjugated to the one remainingactive cysteine on the FGF2 mutein (FIG. 4). The reaction is monitoredby size exclusion HPLC and the conjugate purified using aheparin-Sepharose column followed by Sephacryl S-100 gel filtration.

[0435]FIG. 4 illustrates a schema for the synthesis and purification ofthe Fab-FGF2 conjugate. It should be expressly understood that thisschema may be applied to the synthesis and purification of anyFab-ligand conjugate and is thus not limited to the one illustrated.

[0436]FIG. 5A shows the results of SDS-PAGE of Fab-FGF2 undernon-reducing conditions. Equal amounts (2 ug) of FGF2 (lane 2), Fab(lane 3), or Fab-FGF2 (lane 4) are applied to the gel and compared tothe molecular weight standards (lane 1, in thousands) by staining withCoomassie blue. In FIG. 5B, Western blot analysis of Fab-FGF2 conjugateis shown. The protein is transferred to a nitrocellulose membrane,probed with an anti-FGF2 rabbit polyclonal antibody and then with¹²⁵I-Protein A and visualized by autoradiography. A band is observed forFGF2 (lane 5) and for the Fab-FGF2 conjugate (lane 7), but not for Fabantibody alone (lane 6).

[0437] Integrity of the conjugate is confirmed by SDS-PAGE analysis(FIG. 5A, Lane 4) in which the Coomassie stain showed bandscorresponding to the Fab-FGF2 conjugate and did not show the presence offree FGF2. In addition, Western analysis using an antibody generatedagainst FGF2 showed bands corresponding to the size of the Fab-FGF2conjugates (FIG. 5B, lane 7) and did not show the presence of free FGF2.Thus, it is confirmed that FGF2 is conjugated to the Fab and that excessFGF2 had been removed.

[0438] 2. Functional Validation of Fab-FGF2 Conjugate

[0439] The Fab-FGF2 conjugate is compared with unconjugated FGF2 in anendothelial cell proliferation assay. The results of the endothelialcell proliferation assay showed the conjugation of the Fab to FGF2 didnot interfere with the ability of FGF2 to bind to its cognate receptorand stimulate proliferation (FIG. 6A).

[0440] To determine if the Fab-FGF2 conjugate still retainedknob-binding ability, an ELISA assay is performed. The plates are probedusing an anti-FGF2 primary antibody and then with a goat anti-rabbitsecondary antibody prior to addition of the substrate for visualization.The Fab-FGF2 conjugate had an absorbance of 3.70+0.13 when added towells containing Ad5 knob, compared to 0.35+0.04 in wells without theAd5 knob (FIG. 6B). In addition, the absorbance of the conjugate issignificantly greater than when FGF2 alone is added (1.55±0.03)(p<0.002). Therefore, we are able to show that the Fab portion of theFab-FGF2 conjugate bound to Ad5 knob after conjugation and that the FGF2portion of the conjugate is still functional as evidenced by theendothelial cell proliferation assay.

[0441] As summarized briefly above, the results of the assays areillustrated in FIGS. 6A and 36B. FIG. 6 shows functional validation ofthe Fab-FGF2 conjugate. In FIG. 6A, stimulation of endothelial cellproliferation by FGF2 and the Fab-FGF2 conjugate is shown. Bovine aorticendothelial cells are treated with various concentrations of FGF2 orFab-FGF2 (30 pg/mL to 6 ng/mL) and the cell number determined. Cellcount (×1000) is plotted on the vertical axis, while pg/mL are plottedon the horizontal axis. Open circles represent FGF2, while closedcircles represent Fab-FGF2.

[0442] In FIG. 6B, Fab-FGF2 binding to Ad5 knob in an ELISA isillustrated. Recombinant Ad5 knob is probed with either Fab-FGF2, ablank control, or FGF2. As a control, Fab-FGF2 is added to plates thatdid not contain Ad5 knob. Absorbance is plotted on the vertical axis,while the following are shown on the horizontal axis of the bar graph,proceeding from left to right: No knob+Fab-FGF2; Knob+Fab-FGF2; Knobalone; and Knob+FGF2.

[0443] 3. In Vitro Infection Using Fab-FGF2 Conjugate

[0444] Having shown that the Fab-FGF2 conjugate stimulated endothelialcell proliferation and bound to the adenoviral fiber knob, the conjugateis then used to show targeting of AdCMVLuc to high-affinity FGFreceptors in vitro. Four cell lines—3T3, PANC-1, SKOV3.1p1, andD54MG—are infected with either AdCMVLuc alone, AdCMVLuc premixed withthe Fab, AdCMVLuc premixed with the Fab-FGF2, or AdCMVLuc premixed withthe Fab-FGF2 and the anti-FGF2 antibody.

[0445] Twenty-four hours post-infection, a luciferase assay isperformed. The 3T3, PANC-1, SKOV3.1p1, and D54 MG cells infected withAdCMVLuc alone resulted in luciferase activity of 3.7×10⁴, 5.8×10⁴,8.4×10⁴, and 2.0×10⁶ RLU/ug of protein, respectively (FIG. 7).

[0446]FIG. 7 illustrates the results of in vitro infection of a panel ofcell lines using the Fab-FGF2 conjugate. In FIG. 7A, inhibition ofluciferase expression by the Fab is shown. The four cell lines areinfected with either the AdCMVLuc or the AdCMVLuc premixed with the Fabas described in the text. The data are expressed as a percentage of theluciferase expression when AdCMVLuc alone is used for each cell line.Percentage is plotted on the vertical axis; cell lines 3T3, PANC-1,SKOV3.1p1, and D54 MG are illustrated along the horizontal axis. Openbars represent AdCMVLuc, while closed bars represent AdCMVLuc+Fab.

[0447] In FIG. 7B, luciferase expression in the four cell lines wheninfected with either AdCMVLuc or the AdCMVLuc-Fab-FGF2 conjugate isshown. The bars illustrate the luciferase expression in relative lightunits (RLU) per microgram of protein and represent triplicatemeasurements±standard deviation. RLU/ug of protein is plotted on thevertical axis. On the horizontal axis, cell lines 3T3, PANC-1,SKOV3.1p1, and D54 MG are illustrated. Closed bars represent AdCMVLuc,while cross-hatched bars represent AdCMVLuc+Fab-FGF2.

[0448] In FIG. 7C, inhibition of luciferase expression by the anti-FGF2antibody is shown. The four cell lines are infected with either AdCMVLucpremixed with the Fab-FGF2 conjugate or AdCMVLuc premixed with theFab-FGF2 conjugate and the anti-FGF2 antibody as described above. Thedata are expressed as a percentage of the luciferase expression seenwhen the ADCMVLuc-Fab-FGF2 complex is used for each cell line.Percentages are plotted on the vertical axis; cell lines 3T3, PANC-1,SKOV3.1p1, and D54 MG are illustrated along the horizontal axis. Lightlyshaded bars represent AdCMVLuc+Fab-FGF2, while the dark, closed barsrepresent AdCMVLuc+Fab-FGF2+anti-FGF2 Ab.

[0449] The infection is inhibited in each of the cell lines by 97.0,98.8, 69.3, and 98.3%, respectively, when AdCMVLuc is premixed with theFab antibody (FIG. 7A). Interestingly, each of the cell lines exhibiteda higher level of luciferase activity (2.9×10⁵, 1.3×10⁵, 2.0× 1 0 ⁶, and4.1×10⁶ RLU/ug of protein, respectively) when infected with AdCMVLucpremixed with the Fab-FGF2 conjugate than when infected with AdCMVLucalone (FIG. 7B). The anti-FGF2 antibody inhibited cell infection by theAd-Fab-FGF2 complex by 96.1, 96.3, 90.1, and 94%, respectively (FIG.7C). These results demonstrated that the complex specificallyre-targeted Ad to high-affinity FGF receptors. By re-targeting throughthis pathway, higher levels of gene transfer are achieved than when Adis routed through its native receptor pathway.

[0450] 4. In Vivo Infection Using Fab-FGF2 Conjugate

[0451] Therapeutic index in gene therapy approaches is frequentlydictated by the differential between tumor and non-tumor celltransduction. We thus explored the capacity to achieve tumor-specificdelivery in a murine model of ovarian carcinoma. Luciferase activity inthe tumor and dorsal mesothelial lining in athymic nude mice bearingSKOV3.1p1 tumors in the peritoneum is determined 48 hr after i.p.administration of AdCMVLuc alone, AdCMVLuc premixed with Fab, AdCMV Lucpremixed with Fab-FGF2, or AdCMVLuc premixed with Fab-FGF2 and theanti-FGF2 antibody (Table 1). Table 1 shows luciferase expression(RLU/ug) in the tumor and dorsal mesothelial lining 48 hr afteradministration of the various AdCMVLuc complexes for each athymic nudemouse. TABLE 1 AdCMVLuc + Ab AdCMVLuc + AdCMVLuc + Fab-FGF2 + AnimaladCMVLuc Fab Fab-FGF2 anti-FGF2 # Tumor Lining Tumor Lining Tumor LiningTumor Lining 1 98.2 10.3 180 8.9 5557 1098 1853 26.8 2 36.8 24.2 7.0 8.325,843 452 5578 108 3 133 21.2 11.0 4.5 5633 212 1132 153 4 255 4.5 1777.1 7587 201 294 56.4 5 — — 34.2 33.0 1628 55.0 2648 208 Mean_(—) 13115.1 81.8 12.4 9250 404 2301 110 std dev ±92 ±9.2 ±88.9 ±11.7 ±9525 ±413±2028 ±72.9

[0452] The results illustrated in Table 1-indicate that the tumor ismore susceptible to Ad transduction than the dorsal mesothelial lining(131 vs. 15.1 RLU/ug protein, respectively) and both organs showed areduction in luciferase activity when the Ad-Fab complex is administeredto mice (37.6% inhibition in tumor and 17.9% inhibition in abdominallining). However, when the mice are administered Ad-Fab-FGF2, both thetumor and the abdominal lining exhibited higher luciferase activity(9250 and 404 RLU/ug protein, respectively) than when administered theAd alone. The ratios of luciferase activity in the tumor to theluciferase activity in the dorsal mesothelial lining when AdCMVLuc aloneor AdCMVLuc targeted with Fab-FGF2 are administered are 18.5 and 31.2,respectively. These results demonstrate that the Fab-FGF2 conjugatetargets AdCMVLuc preferentially to the FGF receptor-positive tumor cellsin this in vivo model of ovarian cancer.

Example 3 Preparation of FGF Muteins

[0453] As disclosed above, FGF-related molecules, including analogs,derivatives, fragments, and mimics thereof, are useful in conjugates,compositions, systems and methods of the present invention. Proceduresfor the preparation of FGF muteins are provided hereinbelow for purposesof example and illustration of some of the molecules that are useful asdisclosed herein.

[0454] E. Materials and Methods

[0455] 1. Reagents

[0456] Plasmid pFC80, containing the FGF2 coding sequence, is a gift ofDrs. Paolo Sarmientos and Antonella Isacchi of Farmitalia Cargo Erba(Milan, Italy). Plasmid pFC80, has been described in the PCT ApplicationSerial No. WO 90/02800 and PCT Application Serial No. PCT/US93/05702.The sequence of DNA encoding FGF2 in pFC80 is that set forth in PCTApplication Serial No. PCT/US93/05702.

[0457] Plasmid isolation, production of competent cells, transformationand M13 manipulations are carried out according to published procedures(Sambrook et al., Molecular Cloning a Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Purificationof DNA fragments is achieved using the GENECLEAN II kit, purchased fromBio 101 (LaJolla, Calif.). Sequencing of the different constructions isperformed using the SEQUENASE kit (version 2.0) of USB (Cleveland,Ohio).

[0458] 2. Sodium Dodecyl Sulfate (SDS) Gel Electrophoresis and WesternBlotting

[0459] SDS gel electrophoresis is performed on a PhastSystem utilizing20% gels (Pharmacia). Western blotting is accomplished by transfer ofelectrophoresed protein to nitrocellulose using the PhastTransfer system(Pharmacia), as described by the manufacturer. The antisera to SAP andbasic FGF are used at a dilution of 1:1000. Horseradish peroxidaselabeled anti-IgG is used as the second antibody.

[0460] F. Preparation of the Mutagenized FGF by Site-DirectedMutagenesis

[0461] Cysteine to serine substitutions are made byoligonucleotide-directed mutagenesis using the Amersham (ArlingtonHeights, Ill.) in vitro-mutagenesis system 2.1. Oligonucleotidesencoding the new amino acid are synthesized using a 380B automatic DNAsynthesizer (Applied Biosystems, Foster City, Calif.).

[0462] 1. Mutagenesis

[0463] The oligonucleotide used for in vitro mutagenesis of cysteine 78is AGGAGTGTCTGCTAACC, which spans nucleotides 225-241 of FGF2. Theoligonucleotide for mutagenesis of cysteine 96 isTTCTAAATCGGTTACCGATGACTG, which spans nucleotides 279-302. The mutatedreplicative form DNA is transformed into E. coli strain JM109 and singleplaques are picked and sequenced for verification of the mutation. TheFGF mutated gene is then cut out of M13, ligated into the expressionvector pFC80, which had the non-mutated form of the gene removed, andtransformed into E. coli strain JM109. Single colonies are picked andthe plasmids sequenced to verify the mutation is present. Plasmids withcorrect mutation are then transformed into the E. coli strain FICE 2 andsingle colonies from these transformations are used to obtain the mutantbasic FGFs. Approximately 20 mg protein per liter of fermentation brothis obtained.

[0464] 2. Purification of Mutagenized FGF

[0465] Cells are grown overnight in 20 ml of LB broth containing 100μg/ml ampicillin. The next morning the cells are pelleted andtransferred to 500 ml of M9 medium with 100 μg/ml ampicillin and grownfor 7 hours. The cells are pelleted and resuspended in lysis solution(10 mM TRIS, pH 7.4, 150 mM NaCl, lysozyme, 10 μg/mL, aprotinin, 10μg/mL, leupeptin, 10 μg/mL, pepstatin A, 10 μg/mL and 1 mM PMSF; 45-60ml per 16 g of pellet) and incubated while stirring for 1 hour at roomtemperature. The solution is frozen and thawed three times and sonicatedfor 2.5 minutes. The suspension is centrifuged; the supernatant savedand the pellet resuspended in another volume of lysis solution withoutlysozyme, centrifuged again and the supernatants pooled. Extract volumes(40 ml) are diluted to 50 ml with 10 mM TRIS, pH 7.4 (buffer A). Poolsare loaded onto a 5 ml Hi-Trap heparin-Sepharose column (Pharmacia,Uppsala, Sweden) equilibrated in 150 mM sodium chloride in buffer A. Thecolumn is washed with 0.6 M sodium chloride and 1 M sodium chloride inbuffer A and then eluted with 2 M sodium chloride in buffer A. Peakfractions of the 2 M elution, as determined by optical density at 280nm, are pooled and purity determined by gel electrophoresis. Yields are10.5 mg of purified protein for the Cys⁷⁸ mutant and 10.9 mg for theCys⁹⁶ mutant.

[0466] The biological activity of [C78S]FGF and [C96S]FGF is measured onadrenal capillary endothelial cells in culture. Cells are plated at3,000 per well in a 24 well plate in 1 ml of 10% calf serum-HIDMEM.Cells are allowed to attach, and samples are added in triplicate at theindicated concentration and incubated for 48 h at 37° C. An equalquantity of samples is added and further incubated for 48 hr. Medium isaspirated; cells are treated with trypsin (1 ml volume) to remove cellsto 9 ml of Hematall diluent and counted in a Coulter Counter. Theresults show that the two mutants that retain virtually completeproliferative activity of native basic FGF as judged by the ability tostimulate endothelial cell proliferation in culture.

Example 4 Efficacy and Toxicity of FGF-AD

[0467] As disclosed above, viral vectors re-targeted with polypeptidesthat target the FGF receptor—including derivatives and fragments of FGFand polypeptide portions thereof—are useful in conjugates, compositions,systems and methods of the present invention. Procedures and exemplarydata illustrating some of the novel and unexpected advantages of the useof the constructs of the present invention are provided hereinbelow forpurposes of example and illustration.

[0468] A. Methods

[0469] 1. Toxicity Assessment In Vivo

[0470] Toxicity of FGF Ad βgal and Ad βgal is assessed in female C57B1/6mice (n=5/group). For preparation of FGF Ad βgal or Ad βgal, 77 μg ofFGF-Fab, or an equivalent volume of 0.9% NaCl is incubated for 30minutes at room temperature with 2×10¹⁰ pfu of Ad βgal. 2×10¹⁰ pfu ofeither Ad βgal or FGF— Ad βgal are injected intravenously per mouse(over a 30 second period) in a final volume of 0.32 ml. Control micereceived 0.32 ml of excipient (25 mM Tris pH 7.5, 100 mM NaCl, 10 mg/mllactose). On day 4 post injection, 2 mice per group are sacrificed.Serum is collected for analysis of serum transaminases and alkalinephosphatase. The liver, lungs, spleen, and kidneys are removed andweighed. A portion of liver and lung are immediately snap frozen inliquid nitrogen, stored at −80° C. and then processed for quantitativeanalysis of β-galactosidase activity. Portions of each organ are snapfrozen in OCT using isopentane precooled with dry ice and stored at −80°C. Another portion of each organ is fixed for 4,, hours at 4° C. in 10%neutral buffered formalin and then embedded in paraffin. On day 7, threemice per group are sacrificed and tissues and serum are processed in thesame manner.

[0471] 2. β-Galactosidase Activity Measurement

[0472] β-gal activity is quantitated in mouse liver homogenatesaccording to standard techniques. Briefly, frozen tissues are mincedwith razor blades and homogenized on ice in cold lysis buffer by handusing glass douncers. 0.1 g of organ weight is added per mL of 0.2%Triton, 100 mM Potassium Phosphate lysis buffer, pH 7.8. Homogenates areclarified by two centrifugation steps of 20 minutes each at 4° C. in amicrofuge at 12,000×g. Supernatants are treated with Chelex-100 resin(BioRad catalog # 142-2842) by adding 0.25×volume chelator to eachsample. Homogenates are then vortexed briefly, incubated at roomtemperature for 2-5 minutes, and centrifuged for 30 seconds in amicrofuge at 12K×g. A two-fold dilution series of each supernatant isassayed using the Clontech Luminescent β-gal Detection Kit II (catalog #K2048-1). 10 μl of each sample dilution is incubated with 75 μl ClontechB-gal Reagent in 96-well plates at room temperature for 1 hour and readin a Dynatech Laboratories ML3000 Microtiter plate luminometer. Theactivity of each sample is determined by extrapolation from a standardcurve of β-gal enzyme supplied with the Clontech kit, and is expressedin mU/g organ weight.

[0473] 3. Histological Determination of β-galactosidase Activity

[0474] Eight micron cryostat sections are fixed in 2% PFA, 0.5% GA inPBS pH 7.4 for 30 min. at room temperature. Tissue sections are thenrinsed in PBS containing 0.03% NP-40 and 2 mM MgCl₂ and incubated inX-Gal solution for 16 hr. at 37° C. (1 mg/ml X-Gal, 5 mM K₃Fe(CN)₆, 3 mMK₄Fe(CN)₆ in PBS pH 7.4 containing 2 mM MgCl₂ and 0.03% NP-40). Slidesare rinsed in PBS, postfixed in 10% buffered formalin, counterstainedfor 15 sec. with Nuclear Fast Red, dehydrated and mounted. Formorphological studies, routine hematoxylin and eosin staining isperformed on paraffin embedded tissues.

[0475] 4. In Vivo Tumor Model

[0476] FGF-Ad_(HSVTK) is prepared by mixing 0.3 μg of FGF-Fab with1×10⁸pfu of FGF-Ad_(HSVTK) and incubating for 30 minutes at roomtemperature. Either FGF-Ad_(HSVTK), Ad_(HSVTK), or 20 mM HEPES bufferare then mixed with B16 melanoma cells in suspension at an MOI of 50.This mixture is incubated at room temperature for one hour. Female BDF1mice (n=8/group) had 2×10⁶ B16 cells, treated with eitherFGF-Ad_(HSVTK), Ad_(HSVTK), or 20 mM HEPES buffer implantedintraperitoneally on day 0. Mice are then treated with ganciclovir (orH₂O) beginning on day 1, qdx14, at a dose of 100 mg/kg. Mice are thenfollowed for survival. Statistical analysis is performed usingKaplan-Meier and a Logrank (Mantel-Cox) post-hoc analysis.

[0477] B. Results

[0478] 1. Toxicity Analysis

[0479] To accomplish retargeting of Ad, we have made a bi-functionalmolecule by conjugating FGF-2 to a blocking anti-adenoviral knob Fab.This molecule is then incubated with Ad prior to transduction of cellsin vitro or use in vivo. To determine if FGF-2 retargeted Ad blocks thenative tropism of Ad for the liver, FGF-2Adβgal and Ad βgal are injectedintravenously into mice and expression of βgal in hepatocytes isassessed.

[0480] FIGS. 8A-C illustrate the expression of β-galactosidase in theliver of mice after treatment with Adβgal or FGF2-Adβgal. In Fig. A, noXgal stained cells in the liver of-C57B1/6 mice treated with excipientare seen. In FIG. 8B, numerous Xgal stained hepatocytes are present inthe liver of C57B1/6 mice treated with Adβgal at a dosage of 2×10¹⁰ pfuper mouse, i.v. In FIG. 8C, treatment with FGF2-Adβgal at 2×10¹⁰ pfu permouse, i.v. transduces very few hepatocytes.

[0481] On day 4 post-administration, numerous Xgal stained hepatocytesare present in the livers of mice treated with Adβgal (see FIG. 8B). Inthe livers of mice treated with FGF-2Adβgal, there is a demonstrabledecrease in Xgal stained hepatocytes (FIG. 8C). Quantitation ofβ-galactosidase activity in liver (Table 2) demonstrated 30-fold lessPgal in the FGF-2Adβgal treated group than the in Adβgal treated group.Results are similar on day 7 post-administration for both Xgal stainingand quantitation of β-galactosidase activity (Xgal staining omitted,quantitation in Table 2). TABLE 2 Quantitation of β-Galactosidase in theLiver of Mice Treated with AdβGal or FGF-AdβGal Mean βGalactosidaseActivity (mU/gram)* Treatment Day 4 Day 7 AdβGal 2008, 6542 1719, 50, 91FGF-AdβGal 157, 126 5, 7, 4

[0482]FIG. 9 shows the serum transaminase and alkaline phosphataselevels in mice treated with Adβgal or FGF2-Adβgal. Serum transaminases(AST, ALT) and alkaline phosphatase are measured on day 7 in C57B1/6mice after treatment with either excipient; Adβgal, 2×10¹ pfu, i.v.; orFGF2-Adβgal, 2×10¹⁰ pfu, i.v.

[0483] On day 7 post-administration, serum transaminase levels areelevated 8.2 to 13.6-fold in the Adβgal treated group but only amoderate 3.2 to 4.7-fold in the FGF-2Adβgal treated group (see FIG. 9).Serum alkaline phosphatase is also elevated in the serum of Adβgaltreated mice but is normal in FGF-2Adβgal treated mice.

[0484]FIGS. 10A and 10B illustrate the histopathology of the liver ofmice after treatment with Adβgal or FGF2-Adβgal. Hematoxylin and eosinstained paraffin sections of the liver of C57B1/6 mice treated witheither Adβgal, 2×10¹⁰ pfu, i.v. (FIG. 10A); or FGF2-Adβgal, 2×10¹⁰ pfu,i.v. (FIG. 10B). Extensive hepatocellular necrosis and inflammatoryinfiltrate present in the liver of mice treated with Adβgal. There isnearly complete abrogation of hepatocellular necrosis in the liver ofmice treated with FGF-2Adβgal. Also, very little inflammatory infiltrateis observed.

[0485] Histopathology on day 7 also revealed evidence of significanthepatocellular necrosis and inflammatory infiltrate in the liver of micetreated with Adβgal but analysis of livers from the FGF-2 Adβgal treatedgroup revealed that the hepatocellular necrosis is almost completelyabrogated and no inflammatory infiltrate is present (FIG. 10).

[0486] 2. Ex Vivo Transduction of B16 Melanoma

[0487] To determine whether FGF-Ad can transduce cells which areinsensitive to Ad, the B16 murine melanoma cell line is chosen as thetarget. B16 cells are incubated for 1 hour ex vivo with either Adtk orFGF-2Adtk prior to implantation intraperitoneally in BDF 1 mice.Ganciclovir therapy is initiated in vivo, one day post tumor cellinoculation.

[0488]FIG. 11 shows a survival analysis of tumor bearing mice treatedwith either Adtk or FGF2-Adtk. B16 melanoma cells are treated ex vivofor one hour with either Adtk or FGF2-Adtk and then implantedintraperitoneally into BDF1 mice at 2×10⁶ cells per mouse. Mice are thentreated with either ganciclovir (GCV) or H₂O (as a control) for 14 days,i.p. Survival of tumor bearing mice treated with FGF2-Adtk and thenadministered ganciclovir is prolonged; such mice have a statisticallyprolonged survival compared to all other groups (p<0.001).

[0489] The survival of mice bearing B16 melanoma treated with Adtk plusganciclovir is indistinguishable from the control mice which receiveduntreated B16 tumor cells plus the ganciclovir regimen (FIG. 11). Instriking contrast, mice which received B16 melanoma treated withFGF2-Adtk demonstrated a 247% increase in median survival compared withcontrol mice (FIG. 11).

Example 5 FGF2 Enhancement of Ad-Mediated Delivery of the HSVTK Gene ina Murine Model of Ovarian Cancer

[0490] In a murine model of human ovarian carcinoma, an FGF2-redirectedadenoviral vector carrying the gene for herpes simplex virus thymidinekinase (AdCMVHSV-TK) is shown to result in a significant prolongation ofsurvival compared with the same number of particles of unmodifiedAdCMVHSV-TK. In addition, equivalent survival rates are achieved with atenfold lower dose of the FGF2-redirected AdCMVHSV-TK compared with theunmodified vector. These findings suggest that strategies to enhance theefficiency of infection of adenoviral vectors may be of great clinicalutility.

[0491] As described in previous examples, efficient gene delivery can beachieved via the use of tropism-modified Ad vectors specificallyretargeted to receptors other than the primary receptor recognized bythe knob domain of the Ad fiber. By complexing an Ad5 vector with abispecific conjugate (Fab-FGF2), significant enhancement of genedelivery in four Kaposi's sarcoma cell lines has been demonstrated (seeExample 1 above). To further delineate the therapeutic benefit to thisapproach, conjugates are constructed and utilized in a murine model ofhuman ovarian cancer, as further described herein.

[0492] A. Methods

[0493] 1. Cells and Viruses

[0494] The human ovarian carcinoma cell line SKOV3.1p1 is readilyavailable from a variety of sources. Our supply is obtained from JanetPrice (Baylor University, Houston, Tex.) and maintained in Dulbecco'smodification of Eagle's medium (DMEM). 293 cells are purchased from theAmerican Type Culture Collection, Rockville, Md. and maintained inDMEM/Ham's F-12 medium. (See also Graham, et al., J. General Virol. 36:59-72 (1977).) The media are supplemented with 10% heat-inactivatedfetal calf serum (FCS), glutamine (42 mM), penicillin (100 units/ml) andstreptomycin (100 μg/ml) and the cells are propagated at 30° C. in a 5%CO₂ atmosphere. FCS is purchased from HyClone Laboratories, Logan, Utahand media and supplements are from Mediatech, Herndon, Va.

[0495] AdCMVLuc, an E1-, E3-deleted Ad5 vector which expresses fireflyluciferase under the control of the CMV promoter, is provided by R. D.Gerard, University of Texas Southwestern medical Center, Dallas, Tex.(see Herz and Gerard, PNAS USA 90:2812-2816 (1993)). AdCMVlacZ, anE14-deleted Ad5 vector which expresses E. coli β-galactosidase from theCMV promoter is obtained from De-chu Tang (University of Alabama atBirmingham, Ala.). AdCMVHSV-TK has been described previously and is anE1-deleted Ad5 vector which expresses HSV-TK from the CMV promoter (seeRosenfeld et al., Clin. Cancer Res. 1:1571-1580 (1995)). The recombinantadenoviral vectors are propagated on the permissive 293 cell line andpurified according to standard techniques (Graham and Prevec,“Manipulation of Ad Vectors,” in Methods in Molecular Biology Vol 7,Gene Transfer and Expression Techniques, Murray and Walker (eds.),Humana Press, Clifton, 1991, pp. 109-128).

[0496] 2. Generation and Characterization of Fab-FGF2 Conjugate

[0497] The Fab-FGF2 conjugate is constructed by conjugating the Fabfragment of 1D6.14, a neutralizing monoclonal antibody directed againstthe Ad5 knob, with an FGF2 mutein as described elsewhere herein.Analysis by mass spectrometry indicated that the conjugate contained asingle molecule of FGF2 linked to a Fab fragment. The FGF2 component ofthe Fab-FGF2 conjugate retained the ability to bind its cognate receptorand stimulate endothelial cell proliferation. The Fab component of theFab-FGF2 conjugate retained the ability to recognize trimeric Ad5 knob,as determined in an ELISA.

[0498] 3. Assays of Adenoviral Infection in Vitro

[0499] Preliminary experiments are performed as previously describedherein to determine the optimal neutralizing dose of the 1D6.14 Fabfragment. Sixteen hours prior to infection, SKOV3.1p1 cells are seededin 24-well plates at a density of 24,000 cells per well. Increasingdilutions of the Fab fragment are incubated with 10⁸ PFU of AdCMVLuc for30 min at room temperature in a total volume of 20 μl HEPES-bufferedsaline (HBS). The vector is then diluted with DMEMIF-12+2% FCS(infecting medium) and 100 μl of the complexes are added at an MOI of 50PFU per cell to the SKOV3.1p1 cells. After incubation for 1 h at 30° C.,the infecting medium is aspirated and replaced with 1 ml ofDMEM/F-12+10% FCS (complete medium). Following incubation for a further24 h at 37° C., the cells are lysed and extracts are assayed forluciferase activity by a chemiluminescent assay (Promega, Madison,Wis.). The protein concentration of the lysates is determined in orderto permit normalization of the data. The lowest dose of Fab whichblocked infection is used in subsequent experiments. In addition, sincethe molar ratio of Fab to FGF2 in the conjugate is known to be 1:1, thisvalue is used to calculate the optimal dose of Fab-FGF2 to be employedin subsequent retargeting experiments.

[0500] To determine the ability of the Fab-FGF2 conjugate to enhanceadenovirus-mediated gene delivery, AdCMVLuc (5×10⁷ PFU) is preincubatedwith the optimal dose of the Fab fragment (1.44 μg) or Fab-FGF2conjugate (1.94 μg) in 20 μμl HBS for 30 min at room temperature. Thevector or vector complexes are then diluted in infecting medium and24,000 SKOV3.1p1 cells in 24 well plates are infected at an MOI of 50PFU per cell in a final volume of 100 μl. Inhibition experiments areperformed by adding a polyclonal anti-FGF2 antibody (Sigma, St. Louis,Mo.) to the AdCMVLuc-Fab-FGF2 complex prior to infection. Cell lysatesare assayed for luciferase activity 24 h post-infection. The proteinconcentration of the lysates is determined in order to permitnormalization of the data. Statistical analysis is performed using theStudent t-test.

[0501] In order to quantitate the number of transduced cells, SKOV3.1p1cells are infected with AdCMVLacZ. Sixteen hours prior to infection,SKOV3.1p1 cells are seeded in 6-well plates at a density of 3×10⁵ cellsper well. AdCMVLacZ (5×1⁷ PFU) is preincubated with or without Fab-FGF2(1.94 μg) in 20 μl HBS for 30 min at room temperature. The vector orvector-Fab-FGF2 complexes are then diluted in infecting medium andincubated with the SKOV3.1p1 cells at an MOI of either 5 or 50 PFU percell in a final volume of 200 μl. After 1 h at 30° C., the infectingmedium is aspirated, the cells are washed with PBS and 3 ml of completemedium are added to each well. Expression of β-galactosidase isdetermined 24 h post-infection by staining with the chromogenicsubstrate X-gal according to standard techniques. The cells are rinsedtwice with PBS and fixed with 0.5% glutaraldehyde for 10 min at 37° C.Cells are then washed twice for 15 min with PBS containing 1 mM MgCl₂,after which they are stained with a PBS solution containing 5 mMK₃Fe(CN)₆, 5 mM K₄Fe(CN)₆, 1 mM MgCl₂ and 1 mg/ml X-gal (5-bromo-4chloro-3-indolyl-beta-D-galactoside; Sigma). After removal of the X-galsolution, the cells are overlaid with 70% glycerol, and stored at 4° C.

[0502] 4. In Vivo Survival Experiment

[0503] Female SCID mice aged 6-8 weeks are obtained from the NationalCancer Animal Program (Frederick, Md.), and received an intraperitonealinjection of 2×10⁷ SKOV3.1p1 cells on day 0. In order to study theeffects on survival, the mice are separated into 10 groups of 10animals, except that the control group with tumor cells only contained 5mice. On day 5, the treated groups are injected intraperitoneally with2×10⁸ or 2×10⁹ PFU of AdCMVHSV-TK alone or AdCMVHSV-TK complexed withFGF2. Forty-eight hours later, half of the groups are treated with anintraperitoneal injection of ganciclovir (50 mg/kg bodyweight) for 14days. The mice are monitored daily for survival. Survival differencesbetween control and experimental groups are then compared and thestatistical significance analyzed using the log-rank test.

[0504] B. Results and Discussion

[0505] Binding studies with ¹²⁵I-labeled FGF are first performed inorder to confirm that the target ovarian cancer cell line, SKOV3.1p1,possessed FGF receptors (data not shown). AdCMVLuc, an E1-, E3-deletedAd5 vector which expresses firefly luciferase (Herz, et al., PNAS USA90:2812-16 (1993)), is then premixed with the unconjugated anti-knob Fabfragment or the Fab-FGF2 conjugate prior to infection of SKOV3.1p1 callmonolayers at a multiplicity of infection (MOI) of 50 plaque-formingunits (pfu) per cell. Expression of luciferase activity in infectedcells is determined 24 hours post-infection; this value is directlyproportional to the number of infecting virus particles.

[0506]FIG. 12 illustrates the enhancement of Ad-mediated gene deliveryby the Fab-FGF2 conjugate. AdCNVLuc (5×10⁷ pfu) as preincubated with theoptimal dose of the Fab fragment (1.44 μg) or Fab-FGF2 conjugate (1.94μg) in 20 μL HBS for 30 min at room temperature. The vector or vectorcomplexes are then diluted in DMEM/F-12+2% FCS and 24,000 SKOV3.1p1cells in 24-well plates are infected at an MOI of 50 pfu/cell.Inhibition experiments are performed by adding a polyclonal anti-FGF2antibody (Sigma, St. Louis, Mo.) to the Ad CMVLuc-Fab-FGF2 complex priorto infection. Cell lysates are assayed for luciferase activity 24 hourspost-infection. The protein concentration of the lysates is determinedto permit normalization of the data, which are expressed as relativelight units (RLU) per microgram of cellular protein. Results are themean±SD of triplicate experiments.

[0507] As shown in FIG. 12, when AdCMVLuc is premixed with the Fab-FGF2conjugate the level of luciferase activity is more than 9-fold greaterthan that achieved by the unmodified vector (p<0.0007). This enhancementof infection is specifically mediated by FGF2; gene delivery by theAd-Fab-FGF2 complex is inhibited by anti-FGF2 antibody.

[0508] We next sought to investigate whether this FGF2-mediatedenhancement in gene expression is due to infection of a greaterpercentage of target cells or to more gene copies per transduced cell.SKOV3.1p1 cell monolayers are infected at different MOIs with anE1-deleted Ad5 vector carrying the P-galactosidase reporter gene,AdCMVLacZ, in the presence or absence of Fab-FGF2.

[0509] Histological data indicate that FGF-2 mediated enhancement of Adgene expression is the result of infection of a greater percentage oftarget cells. AdCMVLacZ (5×10⁷ pfu) is preincubated with or withoutFab-FGF2 (1.94 μg) in 20 μL HBS for 30 min at room temperature. Thevector or vector-Fab-FGF2 complexes are then diluted in DMDMIF-12+2% FCSand SKOV3.1p1 cells are infected at an MOI of 5 or 50 pfu/cell.Expression of β-galactosidase is determined 24 hours post-infection bystaining with the chromogenic substrate X-gal. Tissues are examined fromthe following four groups: A: AdCMVLacZ, MOI=5; B: AdCMVLacZ-FGF2,MOI=5; C: AdCMVLacZ, MOI=50; and D: AdCMVLacZ-FGF2, MOI=50 (results notshown).

[0510] Twenty-four hours post-infection, the cells are stained withX-gal in order to demonstrate the expression of α-galactosidase. It isfound that the Fab-FGF2 conjugate mediated Ad infection of a greaterpercentage of target cells than the native virus, permitting thetransduction of a given number of target cells to be achieved by a lowerdose of virus (results not shown).

[0511] It is well recognized that adenoviral vectors produce adose-dependent inflammatory response in rodents and primates.Vector-associated toxicity has also been observed in human clinicaltrials and threatens to prevent the adenovirus from realizing its fullpotential as a vector for human gene therapy. This suggests that itwould be advantageous to reduce the number of Ad particles required fora given level of gene transfer in vivo. Therefore, we sought todetermine whether Fab-FGF2-mediated enhancement of Ad infection could beexploited for therapeutic advantage.

[0512] A murine model of human ovarian cancer is established asdescribed previously by intraperitoneal injection of SCID mice withSKOV3.1p1 cells (Yu, et al., Cancer Research 53:891-8 (1993); Rosenfeld,et al., J. Molec. Med. 74:455-46291996)). Five days later, the treatedmice are injected intraperitoneally at two MOIs (2×10⁸ r 2×10⁹ pfu)either with AdCMVHSV-TK, an El-deleted Ad5 vector which expresses theprodrug-activating HSV-TK gene, or with AdCMVHSV-TK premixed with theFab-FGF2 conjugate. Mice are then treated for 14 days with 50 mg/kg ofthe prodrug ganciclovir (GCV) or with an equivalent volume of serum-freemedium. Ten animals are studied in each group. These animals aremonitored daily and the length of survival of each mouse is recorded(FIG. 13).

[0513]FIG. 13 illustrates the results of FGF2-enhancement of Ad-mediatedexpression of the HSV-TK gene, which augments therapeutic benefit in asurvival experiment. A total of 95 female SCID mice aged 6-8 weeks areinoculated intraperitoneally with 2×10⁷ SKOV3.1p1 cells on day 0. On day5, some mice are injected intraperitoneally with 2×10⁸ or 2×10⁹ pfu ofAdCMVHSV-TK alone or AdCMVHSV-TK complexed with FGF2 (n=20 mice pergroup). Forty-eight hours later, half of the mice in each group (n=10)are treated daily with an intraperitoneal injection of ganciclovir (50mg/kg bodyweight) for 14 days. Control groups consisted of mice whichreceived no virus or GCV (n=5) or mice which are treated with GCV only(n=10). The mice are monitored daily for survival. The percentage ofanimals surviving is plotted against the number of days post tumor cellinoculation.

[0514] As expected, no significant increase in duration of survival overthe group of untreated mice with tumors is observed for those animalstreated with GCV alone (median survival=32 days). Nor is a survivaladvantage conferred in the absence of GCV by injection of eitherAdCMVHSV-TK or AdCMVHSV-TK premixed with Fab-FGF2. However, whenconsidering treatment with ganciclovir, injection of the mice withAdCMVHSV-TK premixed with Fab-FGF2 is shown to result in a significantprolongation of survival time compared to injection with the same numberof particles of unmodified AdCMVHSV-TK. Thus, when a viral dose of 10⁸pfu is employed, the median survival of mice injected with AdCMVHSV-TKpremixed with Fab-FGF2 is 37 days, compared with 35 days observed forthe native virus (p=0.0025).

[0515] Similarly, at a viral dose of 10⁹ pfu, median survival isincreased from 36 to 44 days when the efficiency of adenoviral infectionis enhanced by Fab-FGF2 (p=0.0070). Of note, equivalent survival ratesare achieved with a ten-fold lower dose of the redirected AdCMVHSV-TKcompared to the unmodified vector (37 days for 108 pfu AdCMVHSV-TKcomplexed with Fab-FGF2 vs. 36 days for 10⁹ pfu AdCMVHSV-TK; p=0.3760).

[0516] The fact that the Fab-FGF2 conjugate enhanced Ad infection bypermitting infection of a greater percentage of cells rather than byproducing more copies of the gene per cells is an important feature ofthis therapeutic modality. It has been reported that the antitumoreffect of the HSV-TK/GCV cannot be augmented simply by increasing theHSV-TK enzyme levels per cell (Elshami, et al., Cancer Gene Therapy 4:213-221 (1997)). In a study by Yee et al. exploring Ad-mediated genedelivery of HSV-TK in a murine ascites model of human breast cancer, athree-fold higher viral dose is employed in an attempt to increasesurvival (Human Gene Therapy 7: 1251-7 (1996)). However, they insteadfound that the higher dose led to substantial toxicity and more deaths.

[0517] In contrast, we have been able to augment the efficiency of theHSV-TK/GCV system by increasing the number of cells expressing theenzyme. These results thus demonstrate that the Fab-FGF2-mediatedenhancement in Ad infection observed in vitro yielded a significanttherapeutic benefit in vivo. Enhanced Ad delivery of the HSV-TK gene tothe ovarian tumor cells resulted in an increase in host survivalcompared to an equal does of native vector. Moreover, the enhanced Adinfection permitted an equivalent therapeutic effect using a ten-foldlower dose of AdCMVHSV-TK. Thus, by permitting therapeuticallysignificant levels of gene transfer while minimizing the toxicityassociated with high numbers of virus particles, the foregoing examplesuggests that strategies to enhance the efficiency of infection ofrecombinant Ad vectors may be of general clinical utility.

Example 6 Assessment of Immunogenicity of Retargeted FGF2-ADV ComplexesCompared to ADV

[0518] Adenoviral vectors have been shown to activate specific immuneresponse. The host immune response is specific to adenoviral proteinincluding the fiber knob protein. FGF2-retargeted Ad will be used as astrategy to blunt or block the antiviral immune response.

[0519] To evaluate adenoviral immunogenicity, mice are treated with1×10⁹ pfu of adenovirus alone or FGF2-retargeted adenovirus. The ratioof FGF-Fab to adenovirus is also varied in this experiment. A total offour groups with the total of number of animals in each group at 10 or13. Group 1 animals received 200 μl of excipient (25 nM Tris pH 7.5, 100mM NaCl, 10 mg/ml lactose) given by i.p. administration. Group 2received adenovirus alone at 1×10⁹ pfu in 200 μl i.p. Group 3 receivedFGF2-retargeted adenovirus at 1×10⁹ pfu/200 μl i.p. injection withFGF2-Fab to adenovirus knob ratio of 30:1. Group 4 receivedFGF2-retargeted adenovirus at 1×10⁹ pfu per 300 μl given i.p. with aFGF2-Fab to adenovirus knob ratio of 2000:1. Mice are checked and bodyweights are measured twice weekly.

[0520] On day 21 post i.p. injection, blood samples are collected from 5of the animals from each of the four groups. The blood samples are putinto an Eppendorf tube and allowed to clot. The samples are centrifugedand the serum is collected. Serum samples are assayed by ELISA forproduction of antibodies to adenoviral proteins and specifically toadenoviral knob protein.

[0521] A. ELISA Assays

[0522] 1. Adenoviral ELISA

[0523] Microtiter plates (96 well) are coated with adenovirus (3×10⁸ pfuin 100 μl per well) and incubated overnight at room temperature. Thewells are rinse 3 times with PBS and then blocked with PBS+10% goatserum for 2 hours at room temperature. The wells are rinsed 3 timesfollowed by addition of primary antibody at a dilution of 1:50. After 30minutes at room temperature the wells are rinsed 3 times with PBSfollowed by addition of alkaline phosphatase anti-mouse Ig secondary.After 30 minutes the wells are rinsed with TBS (Tris buffered saline)followed by addition of PNPP substrate. Color reaction is allowed tooccur for 60 minutes.

[0524] 2. Adenoviral Knob Protein ELISA

[0525] Microtiter plates (96 well) are coated with 100 ng of knobprotein in 100 ul per well and incubated overnight at room temperature.The wells are rinsed 3 times with PBS and then blocked with PBS+10% BSAfor 2 hours at room temperature. The wells are rinsed 3 times followedby addition of primary antibody at a dilution of 1:50. After 30 minutesat room temperature the wells are rinsed 3 times with PBS followed byaddition of alkaline phosphatase anti-mouse Ig secondary. After 30minutes the wells are rinsed with TBS followed by addition of PNPPsubstrate. Color reaction is allowed to occur for 60 minutes.

[0526] B. Results

[0527]FIG. 14 illustrates antibody responses at day 21 following asingle injection of excipient, adenovirus or FGF-Fab:Ad conjugate.Optical density (O.D.)×103 is plotted on the vertical axis, while datapoints corresponding to single injections of excipient, Ad, orFGF-Fab:Ad conjugate are identified on the horizontal axis. Opensquares, circles and diamonds correspond to anti-adenovirus proteinresponses, while closed squares, circles and diamonds correspond toanti-knob protein responses.

[0528] As shown, animals treated with excipient had a backgroundresponse ranging from 14 to 43 with a median response of 33 opticaldensity units. Animals treated with adenovirus developed a robustresponse to adenovirus proteins. Response varied from 1180 to 667 with amedian response of 808 optical density units (see FIG. 14). Antibodyresponse generated from the FGF2-retargeted adenovirus (at anFGF2-Fab:Ad ratio of 2000:1) is significantly reduced. Response variedfrom 556 to 38 with a median of 175 optical density units.

[0529] To determine the percentage of response derived from knob proteinthe antisera generated from all the treated groups are analyzed by knobELISA. Animals treated with excipient had background responses varyingfrom 28 to 21 with a median response of 24 optical density units. FIG.14 also shows that animals treated with adenovirus had a significantresponse to knob protein ranging from 582 to 412 with a median responseof 559 optical density units. Antibody response generated from theFGF2-retargeted adenovirus (at a FGF2-Fab: adenovirus ratio of 2000: 1)is significantly reduced. Response varied from 422 to 24 with a medianof 34 optical density units.

[0530] Therefore, it seems clear that retargeting of viral vectors usingpolypeptides reactive with the FGF receptor is a viable strategy, notonly in the context of enhancing delivery and expression of a gene ofchoice, but in reducing the immunogenicity of the viral vector. Suchretargeted vectors may well be more useful in producing systemictherapeutic effects in view of their reduced potential for stimulatingan antibody response in an individual to which such vectors areadministered in a therapeutic context.

Example 7 Enhanced Gene Delivery to Vascular Endothelial and SmoothMuscle Cells

[0531] Based on the rapidly expanding knowledge of the molecular basesof vascular pathology, delivery of therapeutic genes to the vasculatureis a rational approach to the treatment of many diseases. Particularapplications which have been suggested include atherosclerosis, coronaryartery restenosis following angioplasty, peripheral vascular disease andprimary pulmonary hypertension, as well as the neovascularizationassociated with tumor growth (Finkel T, et al., FASEB J. 1995;9(10):843-851; Gibbons GH, et al., Science. 1996; 272(5262):689-693;Nabel E G, Circulation. 1995; 91(2):541-548; Isner J M, et al., Hum GeneTher. 1996; 7(8):989-1011; Isner J M, et al., Lancet. 1996;348(9024):370-374; Rios C D, et al., Arterioscl Thromb Vasc Biol. 1995;15(12):2241-2245; Rodman D M, et al., Am J Respir Cell Mol. Biol. 1997;16(6):640-649; Muller D W, et al., Circ Res. 1994; 75(6):1039-1049).

[0532] The nature of these disorders requires that effective genetherapy strategies must be based on direct in situ gene delivery. Thus,any proposed approach is dependent on a vector vehicle which is capableof achieving adequate gene delivery to target cells in vivo. Of thecurrently available vector systems, the adenovirus has a number ofproperties which make it a promising vector for in vivo applications(Brody S L, et al., Ann N Y Acad. Sci. 1994; 716:90-101) and a number ofgene therapy approaches for vascular diseases have been developed inmodel systems employing these vectors (Rios C D, et al., ArteriosclThromb Vasc Biol. 1995; 15(12):2241-2245; Rodman D M, et al., Am JRespir Cell Mol. Biol. 1997; 16(6):640-649; Muller D W, et al., CircRes. 1994; 75(6):1039-1049; Harrell R L, et al., Circulation. 1997;96(2):621-627; Lemarchand P, et al., Circ Res. 1993; 72(5):1132-1138;Steg P G, et al., Circulation. 1994; 90(4):1648-1656; Rade J J, et al.,Nat Med. 1996; 2(3):293-298; Feldman L J, et al., J Clin Invest. 1995;95(6):2662-2671). In the vasculature however, where there are relativelylow levels of cellular receptors for the adenovirus (Wickham T J, etal., J. Virol. 1996; 70(10):6831-683), the concentration of viralparticles required to achieve high levels of gene delivery is associatedwith a direct cytotoxic effect (Schulick A H, et al., Circ Res. 1995;77(3):475-485; Schulick A H, et al., Circulation. 1995;91(9):2407-2414).

[0533] Because viral toxicity is directly related to the dose of virusused (Schulick A H, et al., Circ Res. 1995; 77(3):475-485; Crystal R G,et al., Nat Genet. 1994; 8(1):42-51), it would therefore be advantageousto achieve an adequate level of transfection with a lower dose of virus.Targeting adenoviral infection to an alternate receptor, which is highlyexpressed on vascular cells, thus appears to be an appropriate methodfor achieving this goal.

[0534] It has been demonstrated hereinabove that the tropism of theadenovirus can be altered using a retargeting strategy. As proof ofconcept, the Fab fragment of a neutralizing antibody against theadenoviral fiber knob domain (Louis N, et al., J. Virol. 1994;68(6):4104-4106; Henry L J, et al., J. Virol. 1994; 68(8):5239-5246)(which binds to the recently identified cellular adenoviral receptor(Bergelson J M, et al., Science. 1997; 275(5304):1320-1323, Tomko R P,et al., Proc Natl Acad Sci USA. 1997; 94(7):3352-3356; Hong S S, et al.,EMBO J. 1997; 16(9):2294-2306)) is conjugated to folate (Douglas J T, etal., Nat Biotech. 1996; 14:1574-1578). This conjugate is then used toretarget adenoviral infection specifically via the folate receptor(Douglas J T, et al., Nat Biotech. 1996; 14:1574-1578).

[0535] A similar strategy is then employed to direct adenoviralinfection to the fibroblast growth factor (FGF) receptor using basic FGF(FGF2) as the targeting ligand (Goldman C K, et al., Cancer Res. 1997;57(8):1447-1451). Using this approach, the transfectability of Kaposi'ssarcoma cells, which possess low levels of adenoviral fiber receptorsbut high levels of FGF receptors, is greatly enhanced. As a rationalextension of this approach, in the present study we chose to use FGF2 asour targeting ligand for vascular gene delivery, taking advantage of theknowledge that vascular cells express FGF receptors (Asahara T, et al.,Circulation. 1995; 92(9 Suppl):11365-371; Sosnowski B A, et al., J BiolChem. 1996; 271(52):33647-33653). In this way, we are able to achievesignificant enhancement of gene delivery to vascular endothelial andsmooth muscle cells, thus enabling a given level of gene expression tobe achieved with a lower concentration of virus particles. Therefore,this strategy may ultimately improve the clinical utility of adenoviralvectors by allowing effective gene delivery in vivo at viralconcentrations below those that result in toxicity.

[0536] A. Methods

[0537] 1. Cell Culture

[0538] Primary cultures of human umbilical vein endothelial cells(HUVECs) are obtained from the laboratory of Dr F. M. Booyse,(University of Alabama at Birmingham, Birmingham, Ala.). These cells areobtained from umbilical cords as previously described (Booyse F M, etal., Blood. 1981; 58:788-796.) and grown on a 1% gelatin coating inMedia 199 (Cellgro, Herndon, Va.) containing 10% heat inactivated fetalbovine serum (FBS; Hyclone Laboratories, Logan, Utah), penicillin (100I.U./mL; Cellgro), streptomycin (69 mmol/L; 100 mg/mL; Cellgro),glutamine (2 mmol/L; Cellgro), heparin (10 U/mL; Elkins-SinnIncorporated, Cherry Hill, N.J.), insulin (1.4 mmol/L; 10 mg/mL),transferrin (0.13 mmol/L; 10 mg/mL) and sodium selenite (0.06 mmol/L; 10ng/mL) (purchased from Becton Dickson Labware (Bedford, Mass.) as ITSstock) and endothelial mitogen (0.1 mg/mL; Biomedical Technologies,Stoughton, Mass.).

[0539] Primary cultures of human coronary artery endothelial cells(HCAECs) are purchased from Clonetics Corporation (Walkersville, Md.)and grown on 1% gelatin coating in EBM-2 media (Clonetics Corporation)containing EGM-2 MV supplements—FBS (5%), hydrocortisone, humanfibroblast growth factor, vascular endothelial growth factor, R3-insulingrowth factor-1, ascorbic acid, human endothelial growth factor,gentamycin and amphotericin.

[0540] Primary cultures of human aortic smooth muscle cells (HASMCs) areobtained from the American Type Culture Collection (Rockville, Md.) andgrown on uncoated flasks in Ham's F12 media (Cellgro) containing 10%heat inactivated FBS (Hyclone), glutamine (2 mmol/L), endothelialmitogen (0.02 mg/mL, Biomedical Technologies), insulin (1.4 mmol/L; 10mg/mL), transferrin (0.13 mmol/L, 10 mg/mL) and sodium selenite (0.06mmol/L, 10 ng/mL) (Becton Dickson Labware).

[0541] All cells are maintained at 37° C. in a humidified atmospherecontaining 5% C0₂.

[0542] 2. Adenoviral Vectors

[0543] A recombinant ElA-deleted adenovirus expressing fireflyluciferase under the control of the cytomegalovirus (CMV) promoter(AdCMVLuc (Herz J, et al., Proc Natl Acad Sci USA. 1993;90(7):2812-2816)) is propagated in the permissive 293 cell line,purified by centrifugation through two cesium chloride gradients andplaque titered on 293 cells by standard techniques (Graham F, et al.,Methods in Molecular Biology. Vol. 7—Gene Transfer and ExpressionTechniques. Clifton, N.J.: Humana Press; 1991:pp. 109-129). Arecombinant ElA-deleted adenovirus expressing the Escherichia coli βgalactosidase gene under the control of the cytomegalovirus promoter(AdCMVLacZ) is prepared as above. An irrelevant virus (AdAmpg, whichencodes the genes for retroviral packaging functions) is used as acontrol in the galactosidase experiments.

[0544] 3. 1D6.14 Fab-FGF2 Conjugate

[0545] The Fab-FGF2 is constructed by conjugating recombinant FGF2(Lappi D A, et al., Anal Biochem. 1993; 212(2):446-451) with the Fabfragment of a neutralizing monoclonal antibody (1D6.14) generatedagainst the adenovirus serotype 5 knob region (Douglas J T, et al., NatBiotech. 1996; 14:1574-1578). The conjugation procedure and subsequentconfirmation of the activity of the Fab and FGF2 components of theconjugate have been described elsewhere (Goldman C K, et al., CancerRes. 1997; 57(8):1447-1451). Briefly, conjugation is performed usingN-succinimidyl 3-(2-pyridyldithio) propionate (SPDP; Pharmacia, Uppsala,Sweden) followed by purification using heparin-Sepharose and SepharoseS-100 column chromatography (Pharmacia). Mass spectrometry of theresulting conjugate indicated a 1:1 molar ratio of Fab to FGF2. Activityof the Fab and FGF2 components is confirmed by enzyme-linked immunoassayand cellular proliferation assay. In brief, ELISA plates are coated withrecombinant adenovirus knob protein, Fab-FGF2 conjugate is applied tothe plates, then bound conjugate is detected using an anti-FGF antibody.FGF activity of the conjugate is confirmed with a proliferation assayusing bovine endothelial cells.

[0546] 4. Adenoviral Infections

[0547] To properly evaluate the effects of FGF2 retargeting, we aimed toconcurrently ablate native viral tropism and redirect infection viaFGF2. Therefore, preliminary experiments are conducted to determine theoptimal Fab-FGF2 to adenovirus ratio which would achieve this. Firstly,the dose of Fab required to block infection via the native receptor isdetermined by titration. The lowest dose of Fab which maximally blockedinfection (implying all Fab binding sites on the virus are occupied) isdetermined by luciferase assay (see below) and chosen as the basis forsubsequent calculations (data not shown). We then used the same molarratio of Fab-FGF2 to virus as the optimized Fab to virus ratio, based onthe fact that the conjugate contained a 1:1ratio of Fab:FGF2. Foranalysis of transfection, cells are harvested by trypsinization,assessed for viability by trypan blue exclusion and plated into 24 wellplates at a density of 24,000 cells per well.

[0548] Twenty four hours later, cells are infected with adenoviralvector. AdCMVLuc (5×10⁶ plaque forming units (pfu) in 1 ml, diluted fromstock in HEPES-buffered saline(HBS; 150 mmol/L HEPES, 20 mmol/L NaCl, pH7.8) is mixed with Fab (0.2 mg) or Fab-FGF2 (0.27 mg) in 1.5 mLpolypropylene microcentrifuge tubes and incubated at room temperaturefor 30 minutes in a total volume of 5 ml. For experiments in whichdifferent concentrations of virus are used, the amount of Fab orFab-FGF2 is adjusted to keep the proportions constant. For blockingstudies, 2 ml of a rabbit polyclonal anti-FGF antibody (Sigma ChemicalCo) or 10 mg of soluble recombinant FGF receptor extracellular domain(Austral Biologicals, San Ramon, Calif.), are added to the tube andfollowed by a further incubation of 30 min at room temperature.

[0549] Just prior to infection, the volume of each mixture is brought to350 ml with warmed (37° C.) DMEM/F12 (50:50) media (Cellgro) containing2% FBS, glutamine, penicillin and streptomycin. Blocking with excessFGF2 is performed by preincubating cells for 30 min with a 100 foldexcess of free FGF2 (compared to the amount in the conjugate) as well asincluding this amount of FGF2 in the infecting media. Blocking withheparin is performed by using a concentration of 500 U/ml in theinfecting media (i.e. 50-fold excess compared to the concentration ofheparin in the HUVEC propagation medium). Complete media are removedfrom the cells and replaced with the virus-containing media, 100 ml perwell in triplicate. Trays are incubated at 37° C. in 5% CO₂ atmospherefor 1 hour, then the infecting media are aspirated, cells are gentlywashed once with Dulbecco's phosphate buffered saline (D-PBS, Cellgro)and 500 ml of the appropriate complete media are added to the wells.Cells are incubated a further 24 hours, then luciferase reporter geneexpression is assayed.

[0550] 5. Luciferase Assay

[0551] Luciferase expression is analyzed using a Luciferase Assay Systemkit (Promega, Madison Wis.), according to the manufacturer'sinstructions. Briefly, media are aspirated from cells, cells are washedwith PBS, then lysed in Promega cell lysis buffer (100 ml per well).Twenty microliters of lysate are added to 100 ml of Promega luciferaseassay reagent and determinations of relative light units (RLU) are madeusing a Berthold luminometer calibrated to ensure the RLU readout iswithin the linear range of the system.

[0552] 6. β-Glactosidase Assay

[0553] For analysis of β galactosidase gene expression, AdCMVLacZ iscomplexed with Fab or Fab-FGF2 in the manner described for AdCMVLuc,then cells are infected as above. Beta-galactosidase activity isassessed 48 hours later. Media are removed from cells and cells arewashed once with PBS, then fixed in 0.5% glutaraldehyde for 10 mins.Following 2 washes with 10 mM magnesium chloride, cells are stainedovernight at room temperature in the dark with a solution containing 1mg/mL X-gal (GibcoBRL, Grand Island N.Y.). Negative controls includedstaining of uninfected cells and staining of cells infected with anirrelevant virus (AdAmpg). The number of stained versus total cells arecounted in three random high power (100×) fields.

[0554] Beta-galactosidase expression is also assessed by fluorescentactivated cell sorting (FACS) analysis. For these experiments, cells areplated at 100,000 cells per well in six well plates, then infected withadenovirus alone or adenovirus-Fab-FGF2 complexes, prepared as above.After 48 hours, cells are harvested by trypsinization, resuspended in asolution of 10 mmol/L HEPES, 4% FCS in D-PBS (referred to as stainingmedium) at 100,000 cells in 100 ml in 6 mL FACS tubes. Cell suspensionsare warmed for 10 minutes at 37° C., 100 ml of 2 mmol/L fluoresceindi-galactopyranoside (FDG; Sigma Chemical Company) is added, then thereaction stopped after one minute with the addition of 500 ml of icecold staining medium, then 500 ml cold 2% paraformaldehyde, followed byanalysis using a Becton-Dickson FACScaliber machine.

[0555] 7. Tritiated Adenovirus Binding Assay

[0556] A binding assay using ³H labeled adenovirus is performed asdescribed (Wickham T J, et al., J. Virol. 1996; 70(10):6831-6838).Briefly, cells are harvested from confluent 80 cm² flasks with Versene(GibcoBRL) and resuspended at a density of 10⁷ cells per mL. ³H-AdCMVLuc(10,000 cpm, specific activity 1.5×10⁻⁵ counts per particle) isincubated with Fab, Fab-FGF2 or Fab-FGF2+anti FGF antibody, as describedabove, then added to 10⁶ cells in a final volume of 200 ml of Dulbecco'sModified Eagle's Media (DMEM; Cellgro), 10 mmol/L HEPES, 1 mmol/Lmagnesium chloride. Cell suspensions are shaken at 4° C. for 1 hour,washed with 4 mL cold D-PBS/0.1% bovine serum albumen then centrifugedat 1500 rpm for 10 minutes The cell pellet is resuspended in 200 mlD-PBS/0.1% bovine serum albumen and transferred into 5 ML scintillationfluid for counting in a scintillation counter (Packard, 1900TR liquidscintillation analyzer).

[0557] 8. FACS Analysis for FGF Receptors

[0558] HUVECs are harvested by trypsinization, washed twice with cold(5° C.) D-PBS, then fixed with 1% paraformaldehyde for 30 minutes onice. Following two washes with cold D-PBS, cells are resuspended inD-PBS (200,000 cells in 100 ml), then a monoclonal antibody against FGFreceptors (EcR6, PRIZM Pharmaceuticals, San Diego Calif.) is added to afinal concentration of 50 mg/mL and incubated for 30 minutes on ice.This antibody is chosen because the epitope it recognizes has beenmapped to a region shared by all four described FGF receptors. Controlsconsisted of mouse IgG (Sigma) and no primary antibody. Following twoD-PBS washes, goat anti-mouse FITC labeled secondary antibody (JacksonImmunoresearch Laboratories Inc., West Grove Pa.) is applied at 1:100dilution in D-PBS, for 30 minutes on ice, then cells are washed twicewith D-PBS, resuspended in 1% paraformaldehyde and analyzed using aBecton-Dickson FACScaliber machine.

[0559] 9. Statistical Analysis

[0560] Comparisons between different vector groups are made using singlefactor ANOVA and Student's t-test, with significance accepted at p<0.05.

[0561] B. Results And Discussion

[0562] 1. FGF2 Retargeting of Adenovirus Enhances Gene Expression inHUVECS

[0563] The adenovirus is a promising vector for in situ gene delivery tothe vasculature. However, the achievement of high levels of transductionof vascular cells in vivo is limited by target cell cytotoxicity at highconcentrations of virus. Therefore, we aimed to develop a strategy whichwould enable a reduction in the concentration of adenoviral vectornecessary to achieve a given level of transfection. Because the level ofnative adenoviral receptors on vascular cells is relatively low, it ishypothesized that enhanced gene delivery could be achieved by targetingadenoviral infection to an alternate receptor, the FGF receptor, whichis expressed on vascular cells. To explore this possibility, HUVECs aretransfected with AdCMVLuc alone or following incubation of the viruswith a retargeting conjugate, which is formed by linking FGF2 to the Fabfragment of a neutralizing antibody directed against the adenoviralfiber knob domain. As a control to confirm binding of the Fab to thevirus, cells are also infected with virus which had been incubated withFab alone. Twenty four hours later, luciferase reporter gene expression,which is proportional to the number of infecting virus particles, isassessed. Transfection with AdCMVLuc is inhibited 87±3% (mean±SD ofthree experiments) by 1D6.14 Fab, thus confirming the stability of Fabbinding to adenoviral knob in these experiments. Transfection with theadenovirus-Fab-FGF2 complex resulted in a significant 32.4±6.6 foldenhancement of luciferase expression compared to infection with virusalone (mean±SD of three experiments. FIG. 15A). The substantialimprovement in gene expression supported our hypothesis thatadenovirally mediated gene delivery could be enhanced by targeting viaFGF2. Further experiments to confirm that this effect resulted fromgenuine retargeting mediated by FGF2 are then conducted.

[0564] 2. Enhanced Gene Expression is Mediated by Increased Binding ofAd to Cells

[0565] To investigate whether the enhancement in adenovirus mediatedgene delivery is specifically mediated by FGF2, the Fab-FGF2 retargetedvirus is incubated with a polyclonal antibody against FGF, an excess offree FGF2, or with heparin (which binds to FGF) prior to infection ofHUVECs. Transfection is inhibited by each of these reagents; 82±4%,41±29%, and 97±0.6% respectively (means±SD of three experiments),confirming that the enhancement is specifically mediated via FGF2 (FIG.155B).

[0566] In a separate experiment, inhibition of 77%±4% (mean±SD oftriplicate determinations) is also seen by incubating theadenovirus-Fab-FGF2 complex with soluble FGF receptor (data not shown).Importantly, neither heparin nor excess FGF2 had any effect ontransfection by adenovirus alone. It is theoretically possible that theenhancement seen with the adenovirus-Fab-FGF2 complex could have beendue to either a change in adenoviral binding, as we proposed, or due toa stimulatory effect of FGF2 per se, although the lack of enhancementwith excess free FGF2 suggested the latter is not the case.

[0567] However, to answer this question more specifically, cells areinfected with adenovirus alone or in the presence of an equimolar amountof free FGF2 to that contained in the dose of conjugate used forretargeting. Results show that this dose of free FGF2 alone had noeffect on adenoviral transduction (FIG. 15C). Therefore, the enhancementseen is not due to a stimulatory effect of FGF2. To confirm that theenhancement of transduction seen with Fab-FGF2 is due to enhancedadenoviral binding, a binding assay using ³H-labeled adenovirus isperformed. This assay is performed using HUVECs harvested from confluent75 cm² flasks. Results show an enhancement of binding of radiolabeledvirus to HUVECs when the virus is complexed to Fab-FGF2 as compared tovirus alone (FIG. 15D). Taken together, these results confirm that theenhancement in gene expression observed is likely due to increasedbinding of adenovirus when retargeted via FGF2. Thus, these findingssupport the hypothesis that the transduction of endothelial cells can beimproved by targeting Ad via a heterologous receptor.

[0568] 3. FGF2 Retargeting Enhances Ad-Mediated Gene Expression inCoronary Artery Endothelial and Vascular Smooth Muscle Cells

[0569] To further explore the potential of FGF2 retargeting ofadenovirus for enhanced vascular gene delivery, and to ensure theeffects we noted are not peculiar to HUVECs, we investigated the effectof FGF2 retargeting on gene delivery to primary cultures of humancoronary artery endothelial cells and human aortic smooth muscle cells.Cells are infected in triplicate as previously described, and threeexperiments are performed for each.

[0570] Transduction of these cells is significantly enhanced byFab-FGF2. Results in the endothelial cells showed an enhancement of4.55+/−1.3 fold (mean+/−SD, p<0.01) compared to adenovirus alone and aneven greater enhancement of 92.6+/−2.6 fold (mean+/−SD, p<0.01) in thesmooth muscle cells. (FIGS. 16A-B). These findings provide additionalevidence that FGF2 retargeting of adenoviral infection is a usefulstrategy to enhance gene delivery to relevant vascular cells.

[0571] 4. FGF2 Retargeting of Adenovirus Allows a Reduction inAdenoviral Dose

[0572] Because our primary goal is to provide a means to reduceadenoviral dose as a way of avoiding cytotoxicity, we next soughtevidence that the enhancement in gene expression seen with FGF2retargeting would enable a reduction in the dose of virus necessary toachieve the same level of transgene expression. HUVECs are infected withAdCMVLuc at a dose of 10, 50 or 100 pfu/cell with or without Fab-FGF2.

[0573] A luciferase assay performed 24 hours later showed anapproximately equivalent level of transduction using 10 pfu/cell withFGF2 retargeting as is seen with 100 pfu/cell when virus alone is used(FIG. 17). We recognized however, that the enhanced luciferase reportergene expression seen with FGF2 retargeting could potentially either bedue to greater expression per cell, or to a greater number of transducedcells, or both. Therefore the number of transduced cells is determinedby infection with AdCMVLacZ with or without Fab-FGF2 and assessingβ-galactosidase expression by staining and counting of cells.

[0574] These experiments demonstrate a greater number of transducedcells for the same dose of virus when using Fab-FGF2 retargeting (notshown). Data gathered using HUVECs indicated that transfection withunmodified adenovirus at a dose of 50 pfu/cell led to a transfectionefficiency of 10% which is increased to 100% with FGF2 retargeting atthe same concentration of virus. For smooth muscle cells, the relativeenhancement is even greater, with a transfection efficiency of <1% bythe unmodified virus increasing to 100% with FGF retargeting.

[0575] Analysis is also performed by assessing transduction withAdCMVLacZ at 10, 50 or 100 pfu/cell with or without Fab-FGF2 followed byFDG staining and FACS analysis. This analysis confirms that FGF2retargeting resulted in both an increase in the number of transducedcells as well as an increase in the amount of expression per cell, evenif 100% transduction had been achieved with adenovirus alone (data notshown). These experiments confirm that a lower dose of virus can achievethe same degree of reporter gene expression when retargeted via FGF2 andillustrate that the effect occurs across a range of viral doses.

[0576] 5. Enhancement of Gene Delivery by FGF Retargeting is Greater inProliferating Cells

[0577] Because we are retargeting to a receptor which is known to beupregulated in the context of proliferation and tissue injury in vivo(Casscells W, et al., Proc Natl Acad Sci USA. 1992; 89(15):7159-7163;Yamada K, et al., Acta Neurochirurgica—Supplementum. 1994; 60:261-264;Lindner V, et al., Circ Res. 1993; 73(3):589-595) and organ culture invitro (Daley S J, et al., Am J Pathol. 1996; 148(4):1193-1202), wewished to assess whether there is any detectable difference between therelative enhancement in gene transfer with FGF2 retargeting inproliferating versus quiescent cells in vitro and whether anydifferences correlated with levels of FGF receptor expression. Wetherefore attempted to downregulate FGF receptor expression bymaintaining HUVECs in confluent culture for 5 or 10 days, then comparedthe effect of FGF2 retargeting in these cells to the results seen withcells which had been in non-confluent culture for 24 hours only (asdescribed above).

[0578] Confluent cultures are prepared by plating cells at 24, 000 perwell in 24 well plates, then allowing them to reach confluence with nofurther feeding (to day 5) or with one change of media only (on day 5for those cells maintained for 10 days). Under these conditions, at 5and 10 days the cells appeared relatively quiescent as evidenced by atotal lack of mitotic figures, but good viability is maintained (goodmorphology, no evidence of cell death). Cells are then infected withAdCMVLuc with or without FGF2 retargeting and fresh complete media isadded to the wells.

[0579] A luciferase assay is performed 24 hours later. Results areexpressed as a ratio of the gene expression seen in the cells infectedwith retargeted adenovirus, compared with cells plated at the same timewhich are infected with unmodified virus (data not shown). In this waywe corrected for any factors which might impact on adenoviraltransduction per se with extended time in culture (e.g. cellularmetabolic rate).

[0580] The data indicate that with progressively longer time in culture,the enhancing effect of FGF2 retargeting is reduced, such that by day10, FGF2 retargeting actually led to a relative reduction of 54+/−6% ingene expression compared to adenovirus alone. Even at this time pointhowever, the level of transduction with Fab-FGF2 retargeting is stillgreater than that seen when infection is blocked with Fab (data notshown), indicating that a degree of FGF2 retargeted infection is stilltaking place.

[0581] Once it became apparent that the FGF2 retargeting strategy ismediated by FGF2 binding to cells, we sought to investigate whether thereduction in enhancement we saw in the confluent cells could beexplained at least in part by a relative reduction in FGF receptorexpression in these cells, as has been reported for quiescent cells invivo (Casscells W, et al., Proc Natl Acad Sci USA. 1992;89(15):7159-7163; Lindner V, et al., Circ Res. 1993; 73(3): 589-595).

[0582] To investigate this, FACS analysis for FGF receptors is performedusing rapidly proliferating cells and cells maintained in confluentculture as above. We used a monoclonal anti-FGF receptor antibody (EcR6)which recognizes a common epitope shared between all four described FGFreceptor subtypes, and negative controls consisting of cells incubatedwith mouse IgG or no primary antibody. Using this technique we are ableto detect a 60% reduction in the proportion of cells staining positivelyfor FGF receptors in the cells maintained at confluence for 10 dayscompared to the non-confluent cells (data not shown). These data thusshow a trend which is consistent with the published in vivo reports andprovide evidence for a correlation between retargeted gene transfer andthe level of expression of the targeted receptor.

[0583] 6. Discussion

[0584] Our established retargeting strategy has been further expanded toachieve an increase in adenoviral mediated gene delivery to vascularcells. The goal of reducing the concentration of virus required for agiven level of transduction has now been achieved. These findings arerelevant to the clinical implementation of gene therapy becauseadenoviral vectors can cause direct cytotoxic effects at high doses(Crystal R G, et al., Nat Genet. 1994; 8(1):42-51). This effect isespecially apparent in the vasculature, where a dramatic fall intransduction efficiency and loss of vascular cells is seen over a fairlynarrow range of viral concentrations (Schulick A H, et al., Circ Res.1995; 77(3):475-485; Schulick A H, et al., Circulation. 1995;91(9):2407-2414). Thus, the approach described in the present exampleholds promise as a means to achieve high transfection efficiencies invivo while avoiding the high doses of virus associated with cytotoxiceffects.

[0585] In addition to showing enhancement of gene expression, we haveinvestigated the mechanism by which this occurred. Enhancement of genedelivery is blocked by an anti-FGF antibody, excess FGF2, soluble FGFreceptor and by heparin. These findings clearly indicate that theresponse is mediated by FGF2. In addition, our results show that theeffect on gene expression is due to an enhancement of binding of virusto cells in the context of FGF2 retargeting, as opposed to any potentialstimulatory effect of FGF2 per se. The results are thus in keeping withour goal of retargeting infection through an alternate receptor. Thisfinding has important practical implications for the potential in vivoutility of this approach. A true retargeting mechanism is much morelikely to be effective in vivo than a mechanism based on FGF2stimulation because FGF2 has an extremely short half life in vivo andgenerally must be given by infusion or in a sustained releaseformulation for stimulatory effects to be seen (Edelman ER, et al., JClin Invest. 1992; 89(2):465-473).

[0586] The binding of FGF2 to cellular receptors is a complex processinvolving high affinity tyrosine kinase receptors (of which four havebeen described) as well as binding to low affinity binding sites(heparan sulfates) on the cell surface (Yayon A, et al., Cell. 1991;64(4):841-848). There is evidence that the biological responses of FGF2are mediated by binding to heparan sulfates initially, then also to thehigh affinity receptor, thus forming a trimeric complex (Roghani M, etal., J Biol Chem. 1992; 267(31):22156-22162). The exact mechanism ofincreased binding of virus to cells with FGF2 retargeting is notimmediately apparent. While a degree of blocking of transduction is seenwith excess free FGF competition, which implies high affinity binding(Roghani M, et al., J Biol Chem. 1992; 267(31):22156-22162), we observedthe most dramatic blocking effect with heparin, which impacts on theinteraction of FGF2 with both high and low affinity binding sites(Guimond S, et al., ^(J Biol Chem.) 1993; 268(32):23906-23914). Thus,although the enhanced viral binding is due to FGF2, the relativecontribution of high and low affinity binding sites to this effect isunresolved.

[0587] As with many growth factor receptors, FGF receptors aredifferentially expressed in quiescent versus proliferating or injuredcells, with upregulation in the latter group (Casscells W, et al., ProcNatl Acad Sci USA. 1992; 89(15):7159-7163; Brothers TE, et al., J SurgRes. 1995; 58(1):28-32; Speir E, et al., J Cell Physiol. 1991;147(2):362-373). Thus, we examined the effect of FGF2 retargeting oncells maintained at confluence for a prolonged period, as well asrapidly proliferating cells. Interestingly, we found that the relativeenhancement seen with FGF2 retargeting decreased with prolonged time inculture, as did the expression of FGF receptors as measured by FACSanalysis. These findings raise the prospect that FGF2 retargeting maypermit a degree of adenoviral vector selectivity for proliferating orinjured cells in vivo. This would be advantageous in a number oftherapeutic situations relevant to cardiovascular medicine as well asthe angiogenesis associated with neoplasia. There is in fact someprecedent for suggesting FGF2 as a selective targeting ligand for sitesof vascular pathology in a study by Casscells et al, where a conjugatebetween FGF and the toxin saporin is used to selectively eliminateproliferating smooth muscle cells in a model of angioplasty restenosis(Casscells W, et al., Proc Natl Acad Sci USA. 1992; 89(15):7159-7163).In this setting, the surrounding quiescent cells are unaffected and theeffects seen correlated with the distribution of FGF receptors asdemonstrated by radioligand binding. FGF2-saporin is subsequently shownto inhibit neointimal formation in an angioplasty model (Farb A, et al.,Circ Res. 1997; 80(4):542-550), and selective targeting has also beendemonstrated in a model of arteriovenous grafts (Chen C, et al.,Circulation. 1996; 94(8):1989-1995). Whether such selectivity will beseen in the context of adenoviral targeting however, awaits in vivoinvestigation.

[0588] The many advantages of the adenovirus vector for in vivo usewhich make it attractive for retargeting strategies have also beenrecognized by other investigators. Wickham et al recently demonstratedenhanced (7-9 fold) luciferase gene delivery to endothelial and smoothmuscle cells in culture using a bispecific anti-FLAG/anti-integrinantibody conjugate and an adenoviral vector with a short fiber and aFLAG epitope engineered into the penton base (Wickham T J, et al., J.Virol. 1996; 70(10):6831-6838). Another strategy developed by this grouptargeted infection to cell surface heparan sulfates using a geneticallymodified virus with polylysine residues at the C-terminal of the knob(Wickham T J, et al., ^(Nat Biotech.) 1996; 14(11):1570-1573). Thesestrategies also hold promise for vascular gene delivery, but in vivostudies are awaited. We have previously co-developed a strategy usingpolylysine in the context of targeted adenovirus-polylysine-DNAcomplexes which achieved efficient targeted gene delivery in vitro(Curiel D T, et al., Hum Gene Ther. 1992; 3(2):147-154). However, the invivo application is limited by complement mediated inactivation of thepolylysine component (Gao L, et al., Hum Gene Ther. 1993; 4(1):17-24).

[0589] The strategy we present herein offers the flexibility to beapplied to any adenoviral serotype 5 vector. In addition, our findingsindicate the possibility of selectivity for proliferating cells based onthe level of FGF receptor expression. In regard to potential in vivoapplication, we have evidence that the binding of adenoviral knob to Fabis stable in the bloodstream (unpublished observations, 1997) and thatFab-FGF2 retargeting can enhance adenovirally mediated gene delivery toperitoneal tumors in a murine model of ovarian carcinoma, which resultsin an enhanced therapeutic effect of a herpes simplex thymidine kinasetransgene (unpublished observations, 1997). Thus, FGF retargeting ofadenoviral vectors holds significant promise for in vivo application.

[0590] Over the last several years, strategies have been described forgene delivery to the vasculature that include the use of specializedcatheters and chemical enhancers (Feldman L J, et al., Gene Ther. 1997;4(3):189-198). The strategy we describe here complements theseapproaches and suggests the possibility of eliciting high levels of geneexpression and transduction efficiency while avoiding direct adenoviralcytotoxicity. This finding has significant implications forcardiovascular disease. In particular, high transduction efficiencieswill be especially useful for the cytostatic strategies currently beingproposed for angioplasty restenosis and other proliferative vasculardisorders. Our approach may also facilitate targeted angiogenic therapyfor myocardial ischemia and peripheral vascular disease, particularly asthere is evidence for upregulation of FGF receptors in the context ofischemia (Yamada K, et al., Acta Neurochirurgica—Supplementum. 1994;60:261-264). Thus, this strategy may have a major impact on commonclinical problems.

Example 8 gene expression is Enhanced in Ad-Infection-Sensitive andAd-Infection-Resistant Cell Lines When Retargeted Ad are used to DeliverTherapeutic Nucleotide Sequences

[0591] As demonstrated in the foregoing Examples and in those to follow,gene expression is enhanced in cell lines to which the retargetedadenoviral vectors of the present invention are administered.Surprisingly, enhanced gene expression was observed not only in thosecell lines understood to be sensitive to adenoviral infection; enhancedexpression was also observed in cell lines that are normally resistantto Ad infection.

[0592] For example, enhanced gene expression is observed when retargetedAd vectors are used in the following cell lines. In each instance, theabbreviation and cell type of each cell line is indicated. Ad-SensitiveCell Lines Panc-1 Pancreatic carcinoma PaCa-2 Pancreatic carcinomaASPC-1 Pancreatic carcinoma BxPC-3 Pancreatic carcinoma Sk-Cha-1Cholangiocarcinoma SKOV3* Ovarian Carcinoma D54MG Glioma ZR-75-1 BreastCarcinoma RW376 Kaposi's Sarcoma CVU-1 Kaposi's Sarcoma

[0593] Ad-Resistant Cell Lines Swiss 3T3 Fibroblast HASMC Smooth MuscleCells HUVEC Endothelial KSY-1 Kaposi's Sarcoma KS-SLK Kaposi's SarcomaKS-1085-1 Kaposi's Sarcoma KS-1085-B Kaposi's Sarcoma B16FO* MelanomaKM12 Colon Carcinoma CT26 Colon Carcinoma OVCAR5 Ovarian Carcinoma K562Myeloid Leukemia

Example 9 Retargeted Ad has Diminished Toxicity and Immunogenicity, andConfers Enhanced Survival in Mice Challenged with Ad-Resistant Tumors

[0594] Adenoviruses (Ad) have been used as vectors to deliver genes to awide variety of tissues. Despite achieving high expression levels invivo, Ad vectors display limitations such as anti-vector immuneresponses, transient expression, and normal tissue toxicity, which limittherapeutic potential. Targeting strategies to abrogate native tropismand redirect Ad uptake through defined receptors should decreasevector-related toxicities, increase transduction efficiency, and thusallow for systemic administration.

[0595] By retargeting Ad using basic fibroblast growth factor (FGF-2) asa targeting ligand, Ad cellular uptake is redirected through FGFreceptors, which are upregulated on diseased or injured cells.FGF-retargeted Ad demonstrates markedly decreased hepatic toxicity,liver transgene expression, and immunogenicity. FGF-retargeting isestablished by conferring sensitivity to tumors that are highlyresistant to Ad infection, resulting in enhanced survival ofAd-resistant tumor-bearing mice. This broadly useful method to redirectnative Ad tropism may offer significant therapeutic advantages.

[0596] Replication-deficient human adenoviruses, mainly serotypes 2 and5, have been used as vectors for gene delivery in a wide variety of celltypes. Despite achieving high expression levels using adenoviralvectors, the toxicity, short-term transgene expression, andimmunogenicity limit the usefulness of adenoviral vectors and haveprevented demonstration of clinical efficacy (Goldman, et al., CancerRes 57, 1447-1451 (1997); Wagner, et al., Annu Rev Med 48, 203-216(1997)). Several approaches are under investigation to either block thenative tropism of adenovirus, decrease its immunogenicity via deletionof parts of its genome, or target the virus to cell types of interest,with mixed success. (See, e.g., Wickham, et al., J Virol 71, 8221-8229(1997); Yang, et al., Proc Natl Acad Sci USA 91, 4407-4411 (1994);Morral, et al., Human Gene Therapy 8, 1275-1286 (1997); Graham andPrevec, Methods in Molecular Biology 109-128 Humana Press, Clifton,N.J., (1991); and Rosenfeld, et al., 1571-1580 (1995).)

[0597] A. Materials and Methods

[0598] 1. Materials

[0599] The FGF2-anti-knob fiber Fab conjugate is made as describedherein (see also Goldman, et al., Cancer Res 57, 1447-1451 (1997)).FGF2-Fab (0.34 mg/mL) is stored at −80° C. in Dulbecco'sphosphate-buffered saline (Gibco BRL, Grand Island, N.Y.). AdCMVHSV-TKhas been previously described and is an E1-deleted Ad5 vector whichexpresses HSV-TK from the CMV promoter. Ad5β-gal is obtained fromMolecular Medicine LLC (La Jolla, Calif.). Ad5β-gal is an E1-deleted,E3-mutated vector which expresses β-gal from the CMV promoter. AC2 cellsare derived from a clone of 293 cells that had been selected for highervirus production levels (Molecular Medicine, LLC).

[0600] Viruses are plaque purified and individual isolates used toinfect AC2 cultures. Virus is purified using chromatographic methods togenerate infectious virus equivalent to CsCl preparation. Particlenumber and plaque titering assays are performed using standard methods².Plaque forming units (pfu) for Ad5HSV-TK and Adβ-gal are determined tobe 4×10¹⁰ per mL and 9×10¹⁰ per mL, respectively. Particle to pfu ratiosfor Ad5HSVtk and Ad5p-gal are determined to be 22.5 and 18.9,respectively.

[0601] 2. Assessment of Hepatic Tropism

[0602] Targeting FGF2-Adβ-gal and Adβ-gal is assessed in female C57BI/6mice. For preparation of FGF2-Adβ-gal or Adβ-gal, 77 μg of FGF2-Fab, oran equivalent volume of 0.9% NaCl, is incubated for 30 minutes at roomtemperature with 2×10¹⁰ pfu of Adβ-gal. On day 0, 2×10¹⁰ pfu of eitherAdβ-gal or FGF2-Adβ-gal are injected intravenously per mouse (over a 30second period) in a final volume of 0.32 mL. This amounts to a 50:1molar ratio of FGF2-Fab to fiber molecules. Control mice received 0.32mL of excipient (25 mM Tris pH 7.5, 100 mM NaCl, 10 mg/mL lactose). Ondays 2, 4, 7 and 12 post injection, 3 to 6 mice per group aresacrificed. Serum is collected for analysis of transaminases andalkaline phosphatase. The liver is removed, weighed, and immediatelysnap frozen in liquid nitrogen, stored at −80° C. and then processed forquantitative analysis of β-galactosidase activity. A portion of liver iseither fixed for 4 hours at 4° C. in 10% neutral buffered formalin andthen embedded in paraffin, or snap frozen in OCT using isopentaneprecooled with dry ice and stored at −80° C.

[0603] 3. Histological Determination of β-Galactosidase Activity

[0604] Eight micron cryostat sections are fixed in 2% paraformaldehyde,0.5% glutaraldehyde in PBS pH 7.4 for 30 min. at room temperature.Tissue sections are then rinsed in PBS containing 0.03% NP-40 and 2 mMMgCl₂ and incubated for 16 hours at 37° C. in 1 mg/mL5-bromo-4-chloro-3-indolylb-D-galactopyranoside (X-Gal) (Fischer), 5 mMK₃Fe(CN)₆, and 5 mM K₄Fe(CN)₆ in PBS pH 7.4 containing 2 mM MgCl₂ and0.03% NP-40. Slides are rinsed in PBS, postfixed in 10% bufferedformalin, counterstained for 15 seconds with Nuclear Fast Red,dehydrated and mounted. For morphological studies, routine hematoxylinand eosin staining is performed on paraffin-embedded tissues.

[0605] 4. Quantitation of β-galactosidase Activity

[0606] β-gal activity is quantitated in mouse liver homogenatesaccording to standard techniques. Briefly, frozen tissues are minced andhomogenized on ice in cold lysis buffer by hand using a glass tissuegrinder. 100 mg of liver weight is added per mL of 0.2% Triton-X, 100 mMpotassium phosphate lysis buffer, pH 7.8. Homogenates are clarified bytwo centrifugation steps of 20 minutes each at 4° C. in a microfuge at12,000 g. Supernatants are treated with Chelex-100 resin (BioRad catalog# 142-2842) by adding 0.25× volume chelator to each sample. Homogenatesare then vortexed briefly, incubated at room temperature for 2 to 5minutes, and centrifuged for 30 seconds in a microfuge at 12,000 g. Atwo-fold dilution series of each supernatant is assayed using theClontech Luminescent β-gal Detection Kit II (catalog # K2048-1). 10 μlof each sample dilution is incubated with 75 μl Clontech β-gal Reagentin 96-well plates at room temperature for 1 hour and read in a DynatechLaboratories ML3000 Microtiter plate luminometer. The activity of eachsample is determined by extrapolation from a standard curve of β-galenzyme supplied with the Clontech kit, and is expressed in mU/g organweight. Statistical analysis of the data is performed using an unpairedt-test.

[0607] 5. Immunogenicity Study

[0608] Female BDF1 mice (n=5/group) are treated intraperitoneally on day0 with 1×10⁹ pfu of Adβ-gal or FGF2 Adβ-gal (at a 2000:1 ratio ofFGF2-Fab to knob monomer). Control mice received 200 μL of PBS. On day21, blood samples are collected and assayed for antibodies by ELISA.

[0609] 6. ELISA Procedures

[0610] Ninety-six well cluster plates (Costar catalog #3590) are coatedovernight with 100 μl per well of either Ad5 (3×10⁸ PFU/well) orpurified fiber protein (0.1 μg/well) diluted in PBS. Plates are thenrinsed three times with PBS and blocked for 2 hours with PBS containing10% goat serum (GIBCO, Grand Island N.Y.). Following three additionalrinses, sera diluted 1:50 in PBS are added as 100 μl volumes andincubated for 30 min. Wells are again rinsed three times with PBS, and100 μl of an optimal dilution of F(ab′)₂ fragments of alkalinephosphatase-labeled goat anti-mouse Ig are added per well. Followingthree rinses in Tris buffered saline (TBS), bound antibody is detectedby the addition of 100 μl of p-nitrophenyl phosphate (Sigma Chemicals,St. Louis Mo.).

[0611] Following a 60 min incubation, substrate reactions are determinedusing a microplate reader set at a wavelength of 490 nm for referenceand 405 nm for detection. All wells are blanked against six wells thathad not received primary antibody, and the mean of three triplicatewells determined for each serum sample. Data are expressed as OD405×10³.

[0612] B. B16 Melanoma Tumor Model

[0613] FGF2-AdHSVTK is prepared by mixing 0.3 or 0.03 μg of FGF2-Fabwith 1×10⁸ pfu of FGF2-AdHSVTK (molar ratio of 3:1 or 30:1 FGF2-Fab toknob) and incubating for 30 minutes at room temperature. EitherFGF2-AdHSVTK, AdHSVTK, or 20 mM HEPES buffer are then mixed with B16melanoma cells in suspension at a multiplicity of infection of 50:1.This mixture is incubated at room temperature for one hour.

[0614] Female BDF1 mice (n=8/group) received 2×10⁶ B16F0 cells (LouWeiner, Fox Chase Cancer Center), treated with either FGF2-AdHSVTK,AdHSVTK, or 20 mM HEPES buffer, implanted intraperitoneally on day 0.Mice are then administered ganciclovir (Cytovene, Roche) (or H₂O)intraperitoneally beginning on day 1, qdxl4, at a dose of 100 mg/kg.Mice are then followed for survival. Statistical analysis is performedusing Kaplan-Meier and a Logrank (Mantel-Cox) post-hoc analysis.

[0615] C. Results and Discussion

[0616] In rodent models, the majority of Ad vector deliveredextravenously is cleared rapidly, within the first 24 hours, in theliver. Concomitantly, there is considerable transduction of liverhepatocytes and associated transgene expression. This is in part due toa high concentration of the Ad cellular receptor, Coxsackie-adenovirusreceptor (CAR), in the rodent liver. Ad transgene expression rapidlydeclines over the first 7 days after Ad vector administration but isassociated with significant liver toxicity as manifest by increasedserum transaminases, necrosis, and inflammation (Yang, et al., Id.(1994); Gao, et al., J Virol 12, 8934-8943 (1996); Hwang, et al., Am JRespir Cell Mol Biol 13, 7-16 (1995)). Retargeting of Ad away from itsnative tropism for CAR may abrogate this liver toxicity.

[0617] We have developed a broadly useful method which retargets Ad byusing a neutralizing Fab to the knob region of the Ad fiber protein. Thefiber protein is used by adenovirus for binding to CAR. By attachingFGF2 (basic fibroblast growth factor) as a targeting ligand to this Fab,this bifunctional molecule targets and redirects adenovirus cellularentry via high affinity FGF receptors. FGF2 binds FGF receptors withunusually high affinity (Kd≈10⁻¹² M) compared to other ligand-receptorinteractions. FGF receptors are upregulated in a number of diseasescharacterized by unwanted cellular proliferation, and many humanmalignancies contain elevated levels of one or more of the fourrecognized FGF receptors.

[0618] We have previously demonstrated that FGF2 targets DNA both invitro and in vivo. Recently, we have demonstrated up to a 12-foldincrease in gene expression using FGF2-retargeted Ad compared to Ad indelivering reporter genes or the HSV thymidine kinase (TK) gene to humanKaposi's sarcoma cell lines in vitro and human ovarian carcinoma cellsboth in vitro and in vivo (Sosnowski, et al., J Biol Chem 271:33647-33653 (1996)). We reasoned that the enhanced in vivo potencyimplied greater specificity, thus it is appropriate to assess whetherFGF2-Ad shows diminished toxicity and immunogenicity by altering itsnative tropism. Redirected tropism may be further evaluated in micechallenged with tumor cells resistant to native Ad infection.

[0619] 1. Redirection of Hepatic Tropism

[0620] To accomplish retargeting of Ad, a bifunctional molecule is madeby conjugating FGF2 to a blocking anti-fiber Fab. This molecule is thenincubated with Ad prior to transduction of cultured cells or use invivo. The high-affinity interaction of this Fab with the knob domain ofthe Ad fiber protein has been measured at 2.1×10⁻⁹ M using Biacoreanalysis. This value is comparable or greater than commerciallyavailable therapeutic antibodies.

[0621] To determine if FGF2 retargeted Ad blocks the native tropism ofAd for the liver, FGF2-Adβ-galactosidase (β-gal) and Adβ-gal areinjected intravenously into mice and expression of β-gal in the liver isassessed. Mice are sacrificed on days 2, 4, and 7 post injection ofeither excipient, Adβ-gal (2×10¹⁰ pfu, i.v.) or FGF2-Adβ-gal (2×10¹⁰pfu, i.v.). Liver tissue is processed and stained with Xgal as describedabove.

[0622] On days 1, 2, 4, 7, and 12 post administration, markedly greaternumbers of Xgal-stained hepatocytes are present in the livers of micetreated with Adβ-gal compared to the livers of mice treated withFGF2-Adβ-gal, which had a profound decrease in X-gal-stained hepatocytes(not shown). No X-gal⁺ hepatocytes are observed in control mice (datanot shown).

[0623] Quantitation of β-galactosidase activity in the liver (Table 3)parallels the histochemical results and demonstrated 12- to 20-fold lessβ-gal in the livers of FGF2-Adβ-gal-treated mice than in the livers ofAdβ-gal-treated mice. By day 12, a modest level of β-galactosidase isstill present in the liver of Adβ-gal-treated mice (259 mU/gram) but isundetectable in the liver of FGF2-Adβ-gal-treated mice. TABLE 3Quantitation of β-galactosidase in the Liver of Mice Treated withAdβ-gal or FGF-Adβ-gal Mean β-galactosidase Activity (mU/gram) TreatmentDay 2* Day 4 Day 7 Adβ-gal 5151* 2668** 804*** FGF2-Adβ-gal 365 217  41 

[0624]FIG. 9 shows the serum transaminase and alkaline phosphataselevels in mice treated with Adβ-gal or FGF2-Adβ-gal. On day 7 postinjection of either excipient, Adβ-gal (2×10¹⁰ pfu, i.v.) orFGF2-Adβ-gal (2×10¹⁰ pfu, i.v.) serum is prepared and transaminases(ALT, AST) and alkaline phosphatase (Alk Phos) are measured. The dataare presented as mean +/−S.E.

[0625] On day 7 post administration, serum transaminase levels areelevated 8- to 16-fold in the Adβ-gal treated group but only 3- to5-fold in the FGF2-Adβ-gal-treated group (see FIG. 9). Serum alkalinephosphatase is also elevated in the serum of Adβ-gal-treated mice but iswithin normal limits in FGF2-Adβ-gal-treated mice.

[0626] Histopathology on day 7 revealed evidence of severehepatocellular necrosis and a marked inflammatory infiltrate in theliver of mice treated with Adβ-gal (Adβ-gal, 2×10¹⁰ pfu, i.v.), butanalysis of livers from the FGF2-Adβ-gal-treated group (FGF2-Adβ-gal,2×10¹⁰ pfu, i.v.) revealed that the hepatocellular necrosis is almostcompletely abrogated and only a minimal inflammatory infiltrate isobserved (data not shown).

[0627] 2. Immunogenicity

[0628] We hypothesized that blocking a potential iunmunodominantepitope, the fiber protein knob domain, would diminish the antibodyresponse to Ad. Accordingly, the humoral response to either FGF2-Ad orAd following a single intraperitoneal injection is evaluated. Serumantibodies directed against total adenovirus or purified fiber proteinare measured by ELISA on day 21.

[0629]FIG. 14 illustrates serum antibody levels of anti-adenovirus andanti-knob protein antibodies in mice treated with either excipient,Adβ-gal, or FGF2-Adβ-gal. On day 21 post injection of either excipient,Adβ-gal (1×10⁹ PFU, i.p.), or FGF2-Adβ-gal, sera are prepared andassayed by ELISA for specific antibody levels directed against eithertotal adenovirus or purified knob protein. Data are presented as themean OD₄₀₅×10³ value of three triplicate wells as determined for eachserum sample. In addition, the arithmetic mean (dashed line) arecompared using one-way analysis of variance and Fisher's procedure forleast significant differences for a posteriori contrasts. Foranti-adenovirus responses, the adenovirus group differs from both theexcipient and FGF2-Ad groups by p<0.0001. For anti-knob proteinresponses, the adenovirus group differs from the excipient group byp<0.0001 and from FGF2-Ad group by p=0.0003.

[0630] Compared to Ad alone, FGF2-Ad induces a lower mean anti-Adantibody response and ⅖ mice had no anti-Ad antibody in the FGF2-Adtreated group. Similarly, mean titers are less, and ⅗ mice generated noantibody response to the knob domain of fiber protein in theFGF2-Ad-treated group compared to the Ad-treated group. The data, whichdemonstrate that the mean anti-knob antibody response is >50% of theanti-Ad response, supports the hypothesis that knob is an immunodominantepitope (see FIG. 14).

[0631] 3. Ex Vivo Transduction of B16 Melanoma

[0632] To determine whether FGF2-Ad can transduce cells which areinsensitive to native Ad infection, the B16 murine melanoma cell line ischosen as the target. B16 tumor cells express FGFR1 and FGFR3 mRNA andare sensitive to FGF2-targeted DNA and protein toxins. B16 cells areincubated for 1 hour ex vivo with either Ad containing the herpessimplex virus thymidine kinase gene (AdHSVTK) or FGF2-AdHSVTK prior toimplantation intraperitoneally in BDF1 mice. Ganciclovir prodrug therapyis initiated in vivo, one day post tumor cell inoculation.

[0633]FIG. 11 shows survival analysis of mice treated with either B16F0tumor cells incubated ex vivo with AdHSVTK or FGF2-AdHSVTK. B16 melanomacells are treated ex vivo for one hour with either AdHSVTK orFGF2-AdHSVTK and then implanted intraperitoneally into BDF 1 mice at2×10⁶ cells per mouse. Mice are then treated with either Ganciclovir orH₂O (as a control) for 14 days, i.p. Survival of tumor bearing micetreated with FGF2-AdHSVTK and then administered ganciclovir have astatistically prolonged survival compared to all other groups (p=0.001).

[0634] The survival of mice bearing B16 melanoma treated with AdHSVTKplus ganciclovir is indistinguishable from the control mice whichreceived untreated B16 tumor cells plus the ganciclovir regimen (mediansurvival 18-19 days; see FIG. 11). In striking contrast, mice whichreceived B16 melanoma treated with FGF2-AdTK, at two different FGF2-Fabto knob molar ratios, demonstrated a2.6-fold increase in median survivalcompared with the control groups (FIG. 11).

[0635] There are several significant obstacles to the use of adenoviralvectors for cytotoxic gene therapy of cancer. First, the transduction ofnormal, non-tumor cells by adenovirus can lead to toxicity which haslimited preclinical studies and initial clinical trials to directinjection into tumors or locoregional delivery to a compartmentcontaining tumor cells (Goldman, et al., Id. (1997); Mazue, et al.,Toxicol Lett 64-65, 329-338 (1992); Ying, et al., Cancer 74, 848-853(1994)).

[0636] Additionally, the immunogenicity of adenoviruses is a potentialhurdle to repeat dosing. We have developed a method to abrogate thenative tropism of adenovirus and redirect its cellular uptake throughFGF receptors. Because there are few normal tissues responsive toadministration of exogenous FGF2 (Wagner, et al., Proc Natl Acad Sci USA89, 6099-6103 (1992); Tomko, et al., Proc Natl Acad Sci USA 94,3352-3356 (1997); Worgall, et al., Human Gene Therapy 8, 37-44 (1997)),transduction of normal tissues with FGF2-Ad should be limited (data notshown).

[0637] Further, redirecting Ad with FGF2 should abrogate the livertropism of adenovirus and decrease its toxicity. Indeed, FGF2-Ad induced12- to 20-fold less transgene expression (β-gal) in the liver thannon-retargeted Ad and had only a modest effect on serum transaminaselevels compared to the robust increase of serum transaminases in themice receiving Ad. When the humoral responses to Ad and FGF2-Ad arecompared, FGF2-Ad displayed reduced immunogenicity, as anti-Ad andanti-fiber protein antibodies are not found in all treated mice, unlikethe Ad-treated group. Although it might be expected that FGF2-anti-fiberFab could block the antibody response to the fiber protein, the bluntingof the response to other epitopes on the virion surface is unexpected.Whether FGF2-Fab is masking these other epitopes through sterichindrance, or whether it directs the clearance of virus through lessimmunogenic pathways, is unknown.

[0638] Multiple doses of the re-engineered vectors of the presentinvention may be administered in vivo without an appreciable level ofhumoral response resulting therefrom. Thus, the modified vectors of thepresent invention are significantly less immunogenic than other vectorsdescribed in the art.

[0639] Furthermore, to demonstrate that native Ad tropism can be fullyredirected to cells bearing FGF receptors, we have shown that anAd-resistant tumor line (B16 murine melanoma) can be made sensitive toFGF2-AdHSVTK transduction. Mice challenged with FGF2-AdHSVTK-treated B16melanoma cells have greatly prolonged survival when compared to micebearing control or AdHSVTK-treated B16 melanoma cells.

[0640] We have also demonstrated FGF2-AdHSVTK to be at least 10-foldmore potent than AdHSVTK in vivo in a human ovarian cancer model whichis sensitive to Ad (Rancourt, et al., Nat Med (1997)). Because of theenhanced efficacy and decreased toxicity of FGF2-retargeted Ad incomparison to Ad, the therapeutic index in vivo is greatly enhanced.FGF2 retargeting of viral vectors thus provides a useful approach totargeted gene delivery, which will be required for successful clinicaloncology applications.

Example 10 Efficacy of Intraperitoneal Delivery of Fgf2Fabad21 in aHer-2/Neu Overexpressing Human Ovarian Carcinoma Model (SKOV31p1)

[0641] An Ad vector that produces an intracellular single-chain antibodyto the Her-2/neu receptor (i.e., an “intrabody”) is evaluated foractivity in vivo. In various cell culture experiments, this Ad (Ad21)has been shown to induce apoptosis in cell lines overexpressing theHer-2/neu receptor. The efficacy of Ad21 and FGF2FabAd21 is tested inthe SKOV31p1 model.

[0642] Methods for the construction of antibodies, includingsingle-chain antibodies, which may be useful as “payloads,” as well assuitable antibodies and fragments thereof that may be used in such atherapeutic context, are available in the art. For example, see U.S.Pat. No. 5,587,458, which describes single-chain antibodies to theHer-2/neu (also known as erbB-2) receptor. The generation and use ofintrabodies is also disclosed in published International App. No. WO96/07321. The disclosures of those documents are incorporated byreference as though fully set forth herein.

[0643] SKOV31p1 cells are implanted ip on day 0. On day 5, mice receivea single ip dose of either Ad21 or FGF2FabAd21 at either of two doselevels and then they are followed for survival. For example, dosages ofAd21 and FGF2FabAd21 administered are 1×10¹⁰ pfu and 5×10⁹ pfu.

[0644]FIG. 21 illustrates the increased survival time seen in an in vivomurine tumor model when an Ad vector re-targeted with FGF2 anddelivering an intrabody payload is administered to SKOV3 tumor-bearingmice. Percent survival is plotted on the vertical axis;post-implantation survival (in days) is plotted on the horizontal axis.Closed circles represent mice receiving Excipient alone (control);closed triangles represent mice receiving non-retargeted Ad deliveringHer-2/neu intrabody; and closed squares represent mice receivingFGF2-retargeted Ad delivering Her-2/neu intrabody. As indicated, N=10;the dose administered was 1×10⁹ ADV or FGF-2 ADV.

[0645] While non-retargeted Ad21 has a minimal effect on survival, inthe high dose FGF2FabAd21 treated group, median survival issignificantly increased (% ILS=128; data not shown). Again, FGF2retargeting of viral vectors shows itself to be useful in both positiveand negative gene therapy contexts and underscores the likelihood thatviral retargeting using polypeptides reactive with pre-selectedreceptors not normally targeted by viral vectors retaining their nativetropism enhances the likelihood of success in a variety of therapeuticcontexts, including clinical oncology applications.

Example 11 Successful Retargeting of Adenoviral Vectors Using KGF and11A8

[0646] In order to demonstrate that a variety of receptor-binding andinternalizing ligands are useful in the retargeting of adenoviralvectors, conjugates of anti-knob Fab and KGF, as well as conjugates ofanti-knob Fab and 11A8 antibody, are constructed as described below.Administration of conjugates retargeted using the aforementionedpolypeptides reactive with FGF receptor demonstrate successfulmodification of Ad tropism as well as a concomitant increase in geneexpression, as described.

[0647] KGF is particularly useful in targeting epithelial cells,hepatocytes, and type II pneumocytes of the lung, which makes it idealfor a variety of gene targeting and delivery applications, as discussedpreviously. Therefore, its incorporation into a ligand-Fab construct andits use as an Ad-retargeting agent provides additional treatmentoptions, particularly when one is addressing disease conditions thatinvolve the cells and receptors specifically targeted by KGF—e.g.hepatocytes and type II pneumocytes.

[0648] A method of generating and purifying Ad knob antigen, which isused to generate anti-knob antibody (from which Fab and other fragmentsare readily prepared) is also described as exemplary.

[0649] A. Purification of Knob Antigen

[0650] A fed-batch fermentation generated approximately 1.4 kg paste andknob is purified using two sequential chromatography steps:cation-exchange followed by immobilized metal ion affinitychromatography (IMAC). Cation-exchange chromatography (CEC) is used as acapture and primary recovery step following lysis and clarification. TheCEC-purified product is then purified by IMAC (charged with nickel)based on the affinity of the poly-histidine n-terminus of knob fornickel. The knob product has been fully characterized (data not shown).

[0651] In the native state, the knob antigen has been shown to exist ashomotrimer. The theoretical molecular weight based on the cDNA whichcodes for knob monomer is 22,539 Da. Based on the analyses to date, thepurified product appears to exist principally as a trimeric moleculewith binding characteristics equivalent to the reference standard. Knobantigen is also useful as an affinity ligand immobilized to achromatographic resin for in-process testing and use in purifyingpreparative quantities of FGF-Fab, KGF-Fab, EGF-Fab and 11A8-Fab.

[0652] B. Preparation of Hybridoma Secreting 11A8 Antibody

[0653] Female Balb/C mice were injected subcutaneously with 10⁷ SK-HEP-1cells in 0.2 ml Dulbecco's PBS to generate the antibody 11A8. Theanimals were reimmunized 14 and 28 days later with 10⁷ cells injectedintraperitoneally. The fusion was done 4 days after the finalimmunization.

[0654] Spleen cells taken from an immunized mouse were fused with NS-0cells using PEG-1500. Hybridoma cells were selected in RPMI-1640containing HAT and 0.005% 2-mercaptoethanol followed by RPMI-1640containing HT.

[0655] An ELISA was used for screening the hybridomas. Briefly, plateswere coated with 50 ul of ECDR 1 (100 ng/ml) overnight at 4° C. Afterwashing, conditioned media samples were added. A second antibodyconjugated to horseradish peroxidase (Bio-Rad, 1:1000 dilution) was usedto detect hybridomas. Cells in positive wells were cloned by limitingdilution.

[0656] Antibodies were purified by ammonium sulfate precipitation andAffi-Gel Protein A agarose column (Bio-Rad, Richmond, Calif.)chromatography according to the manufacturer's protocol. The purity ofthe antibody was checked by a 7.5% PhastGel (Pharmacia, Uppsala, Sweden)under non-reducing conditions with Coomassie blue stain.

[0657] C. KGF-Faband 11A8-Fab

[0658] A preliminary small-scale study is performed in which KGF isconjugated to Fab (anti-knob). The conjugate is purified usingprocedures analogous to those used for the conjugation and purificationof FGF-Fab as described hereinabove (i.e., Heparin-Sepharose affinitychromatography followed by size exclusion chromatography), with minormodifications. In particular, KGF and 11A8 are derivatized with SPDP(monoderivatized) according to the manufacturer's instructions(Pharmacia, Piscataway, N.J.); isolated and then conjugated to Fab. Thefinal bulk conjugate is analyzed by SE-HPLC, and the molar ratios of KGFto Fab are determined by SDS-PAGE/Coomassie (results not shown).

[0659] SE-HPLC demonstrates that the conjugate is heterogeneous but doesnot contain detectable levels of free KGF or free Fab. The molar ratiosof Fab to KGF are estimated at 1:1, based on scanning densitometry ofSDS-PAGE/Coomassie stained gels under reducing conditions (data notshown).

[0660] Biological activity of the KGF component of the conjugate isassessed by a proliferation assay performed on Balb/MK cells. Theconjugate is equipotent to the derivatized KGF and underivatized KGFstandard (not shown). The knob-binding activity and transductionactivity are readily evaluated using standard assays and procedures, asthose of skill in the relevant art will readily appreciate.

[0661]FIG. 20 illustrates the successful retargeting of an Ad vectorlinked to a marker (Adβgal) using either FGF2 or 11A8-Fab and thesuccessful delivery of the marker sequence in HCT116 cells. From left toright, the shaded bars represent Adβgal; Fab; FGF2Fab, 30×; FGF2Fab, 3×;11A8Fab, 30×; and 11A8Fab, 3×. Molar excess of Ligand-Fab:Knob Monomeris indicated in the latter four categories. On the vertical axis, mUPgal/mg protein is indicated. (25K, 72 hr; 300 MOI.)

[0662] The foregoing specification, including the specific embodimentsand examples, is intended to be illustrative of the present inventionand is not to be taken as limiting. Numerous other variations andmodifications can be effected without departing from the true spirit andscope of the present invention.

1 6 1 35 DNA Artificial Sequence Sense Primer 1 atatagaatt ctgtgactactgaggacaca gccac 35 2 35 DNA Artificial Sequence Antisense Primer 2atatacatat gttttttcag ctccagcttg gtccc 35 3 17 DNA Artificial SequenceOligonucleotide 3 aggagtgtct gctaacc 17 4 24 DNA Artificial SequenceOligonucleotide 4 ttctaaatcg gttaccgatg actg 24 5 9 PRT ArtificialSequence Polypeptide capable of targeting receptors such as the CR2receptor 5 Glu Asp Pro Gly Phe Phe Asn Val Glu 1 5 6 11 PRT ArtificialSequence Polypeptide capable of targeting receptors such as the CR2receptor 6 Glu Asp Pro Gly Lys Gln Leu Tyr Asn Val Glu 1 5 10

We claim:
 1. A tropism-modified adenoviral vector system thatspecifically targets cells expressing a preselected receptor,comprising: an antibody or fragment thereof that binds an adenoviralcapsid protein; a targeting ligand that binds the preselected receptor;and an adenovirus containing a nucleic acid molecule that encodes atherapeutic gene product under the control of a promoter; wherein theligand is conjugated to the antibody or fragment thereof and wherein theantibody or fragment thereof is bound to the adenovirus.
 2. The vectorof claim 1, wherein said promoter is a tissue-specific promoter.
 3. Thevector of claim 1, wherein said targeting ligand is a polypeptidereactive with an FGF receptor.
 4. The vector of claim 3, wherein saidpolypeptide reactive with an FGF receptor is an antibody or fragmentthereof.
 5. The vector of claim 4, wherein the antibody is 11A8.
 6. Thevector of claim 3, wherein said polypeptide reactive with an FGFreceptor is selected from the group consisting of FGF-1, FGF-2, FGF-3,FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-11, FGF-13, FGF-14, andFGF-15.
 7. The vector of claim 3, wherein said polypeptide reactive withan FGF receptor is FGF-2.
 8. The vector of claim 3, wherein saidpolypeptide reactive with an FGF receptor is KGF.
 9. The vector of claim1, wherein said targeting ligand is selected from the group consistingof a polypeptide reactive with a VEGF receptor, a polypeptide reactivewith a PDGF receptor, and a polypeptide reactive with an EGF receptor.10. The vector of claim 1, wherein the native tropism of said vector isablated.
 11. The vector of claim 1, wherein the therapeutic gene productis a cytocide or a prodrug.
 12. The vector of claim 1, wherein thetherapeutic gene product enhances cellular proliferation.
 13. The vectorof claim 1, wherein the therapeutic gene product is a biologicallyactive protein or polypeptide that augments or complements an endogenousprotein.
 14. The vector of claim 1, wherein the therapeutic gene productenhances cellular differentiation.
 15. The vector of claim 1, whereinthe therapeutic gene product is a molecule which enhances tissue repairor regeneration.
 16. The vector of claim 1, wherein the therapeutic geneproduct is a molecule which stimulates a protective immune response. 17.The vector of claim 10, wherein the prodrug is thymidine kinase,nitroreductase, or cytosine deaminase.
 18. A pharmaceutical compositioncomprising a physiologically acceptable buffer and a tropism-modifiedadenoviral vector presenting a ligand on its surface, wherein the vectorincludes a nucleic acid molecule encoding a therapeutic gene productunder the control of a promoter.
 19. The composition according to claim18, wherein the ligand is a polypeptide reactive with an FGF receptor.20. The composition according to claim 19, wherein the polypeptidereactive with an FGF receptor is selected from the group consisting ofFGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-11,FGF-13, FGF-14, and FGF-15.
 21. The composition according to claim 19,wherein the polypeptide reactive with an FGF receptor is FGF-2.
 22. Thecomposition according to claim 19, wherein the polypeptide reactive withan FGF receptor is KGF.
 23. The composition according to claim 19,wherein the polypeptide reactive with an FGF receptor is an antibody.24. The composition according to claim 23, wherein the antibody is asingle-chain antibody.
 25. The composition according to claim 18,wherein the ligand is genetically fused with an adenoviral capsidprotein.
 26. The composition according to claim 18, wherein the ligandis chemically conjugated to an adenoviral capsid protein.
 27. Thecomposition according to claim 18, wherein the ligand is conjugated toan antibody or fragment thereof that binds a viral capsid protein. 28.The composition according to claim 18, wherein the therapeutic geneproduct is selected from the group consisting of protein, ribozyme andantisense.
 29. The composition according to claim 18, wherein thetherapeutic gene product is a cytocide.
 30. The composition according toclaim 18, wherein the therapeutic gene product is a prodrug.
 31. Amethod of treating tumors, comprising administering a pharmaceuticalcomposition comprising a physiologically acceptable buffer and atropism-modified adenoviral vector presenting a ligand on its surface,wherein said vector includes a nucleotide sequence encoding atherapeutic gene product under the control of a promoter, wherein saidtherapeutic gene product is selected from the group consisting ofE-cadherin, BGP, Rb, p53, CDKN2/P16/MTS1, PTEN/MMAC1, APC, p331NG1,Smad4, maspin, VHL, WT1, Men1, NF2, MXI1, and FHIT.
 32. A method oftreating ischemia, comprising administering a pharmaceutical compositioncomprising a physiologically acceptable buffer and a tropism-modifiedadenoviral vector presenting a ligand on its surface, wherein saidvector includes a nucleotide sequence encoding a therapeutic geneproduct under the control of a promoter, wherein said therapeutic geneproduct is selected from the group consisting of IGF, TGFβ1, TGF,β2,TGFβ3, HGF, VEGF 121, VEGF 165, FGF1, FGF2, FGF 4, FGF5, PDGF-A, andPDGF-B.
 33. A method of treating connective tissue injury, comprisingadministering a pharmaceutical composition comprising a physiologicallyacceptable buffer and a tropism-modified adenoviral vector presenting aligand on its surface, wherein said vector includes a nucleotidesequence encoding a therapeutic gene product under the control of apromoter, wherein said therapeutic gene product is selected from thegroup consisting of PTH, BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8,BMP10, BMP11, mammalian BMP, and Xenopus BMP.
 34. A method of treatingtissue injury, comprising administering a pharmaceutical compositioncomprising a physiologically acceptable buffer and a tropism-modifiedadenoviral vector presenting a ligand on its surface, wherein saidvector includes a nucleotide sequence encoding a therapeutic geneproduct under the control of a promoter, wherein said therapeutic geneproduct is selected from the group consisting of Bovine VEGF, VEGF,VEGF-B, VEGF-C, Angiopoietin-1, Angiogenin, IGF-1, IGF-II, HGF, PDGF A,PDGF B, TGFB1, TGFB2, and TGFB3.
 35. A method of treating cancer,comprising contacting the cancer cells with a pharmaceutical compositioncomprising a physiologically acceptable buffer and a tropism-modifiedadenoviral vector presenting a ligand on its surface, wherein saidvector includes a nucleotide sequence encoding a therapeutic geneproduct under the control of a promoter, wherein said therapeutic geneproduct is selected from the group consisting of HSVTK, VZVTK,nitroreductase, and cytosine deaminase; and contacting the cancer cellswith a substrate.
 36. The method according to any one of claims 31-35,wherein the ligand is a polypeptide reactive with an FGF receptor. 37.The method according to claim 36, wherein the polypeptide reactive withan FGF receptor is FGF-2.
 38. The method according to any one of claims31-35, wherein the ligand is an antibody or a fragment thereof.
 39. Themethod according to claim 38, wherein the antibody is a single-chainantibody.
 40. The method according to any one of claims 31-35, whereinthe ligand is conjugated to an antibody or fragment thereof that binds aviral capsid protein.
 41. The method according to claim 40, wherein theviral capsid protein is adenovirus fiber protein.
 42. The methodaccording to claim 40, wherein the viral capsid protein is adenovirusknob protein.
 43. The method according to any one of claims 31-35,wherein the ligand is chemically conjugated to a protein on the surfaceof a viral vector.
 44. The method according to any one of claims 31-35,wherein the therapeutic gene product is selected from the groupconsisting of protein, ribozyme and antisense.
 45. The method accordingto any one of claims 31-35, wherein the therapeutic gene product is aprodrug.